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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Rhomboid 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 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 . 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 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. 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 (EGF) recep-

The first members of the rhomboid-like superfamily were iden-

tor [2]. Rhomboid protease homologs have since been found in

tified in [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, -associated rhomboid-like;

pathologies, but their mode of action and druggability are unclear

PD, Parkinson’s disease; RHBDD, rhomboid domain-containing ; RHBDL,

(for more discussion see the article by Lemberg and Adrain in this

rhomboid-like protein; TNF␣, ␣; TACE, TNF␣-converting

; 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.

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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 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 [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 , 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 , 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 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 . 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 [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 [20–23]

pronounced phenotype − muscle wasting and reduced lifespan −

compared to how very widely distributed across 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

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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 −/- 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 [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 , 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 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 (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 , 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 , 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

Another difference of rhomboid mechanism from the mecha- hydrophobic residues in the P4 and P2 positions [70]. This find-

nism of classical serine proteases is that the hydroxyl of the catalytic ing was confirmed by structures of peptidyl chloromethylketone

serine of rhomboid carries out the nucleophilic attack of the prochi- [71] and peptidyl aldehyde [72] inhibitors comprising the P4-P1

ral carbonyl carbon of the scissile bond from its si-face [54,57,58] residues in complex with GlpG (Fig. 2B and C). In addition, E.coli

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Fig. 1. Reaction mechanism of rhomboid proteases.

A) Catalytic reaction cycle of rhomboid protease. A P1–P1 segment of the substrate is shown.

B) Molecular dynamics based model of the scission complex of the P4 to P3 segment of the substrate [71] demonstrates the si-face attack of the prochiral carbonyl carbon

of the substrate by the hydroxyl of the catalytic serine 201 of GlpG. The substrate is shown as green sticks, the enzyme is in grey.

C) Interaction of the oxyanion in the tetrahedral intermediate with the ‘’ in GlpG formed by the side chains of conserved amino acids H150 and N154 and the

main-chain amide of S201 as revealed by the crystal structure of GlpG complexed to diisopropylfluorophosphate (DFP; PDB code 3txt; protein backbone in yellow; ligand in

thin sticks, ligand carbons in yellow) and peptidyl aldehyde (PDB code 5f5b; protein backbone in gray; ligand in thick sticks, ligand carbons in green). The hydrogen bonds

stabilising the oxyanion in the peptidyl aldehyde complex structure are denoted by dashed lines. Note that the position of the oxyanion is nearly identical in both ligands.

D) The detail of the ‘water retention site’ that was proposed to enhance catalytic efficiency of rhomboid protease GlpG [63]. Structure of GlpG apoenzyme (2xov) is shown

as grey surface with the three water molecules (red) proposed to be important for acyl-enzyme hydrolysis located in a shallow cavity near the catalytic dyad (yellow). Part

of the L5 loop overhanging the active site has been removed for clarity. The side-chains shown in green have been proposed to stabilise these water molecules and facilitate

their transfer from the bulk solvent.

GlpG prefers positively charged residues in the P3 and P2 positions ics measurements in lipid membranes revealed that mutations in

[71,72]. The sequence preferences at different positions around the recognition motif of TatA decreased the kcat of AarA by two

the scissile bond, i.e. the recognition motif itself, might thus dif- orders of magnitude with only minor effect on KM [73]. And finally,

fer among rhomboid proteases, and this may contribute to their active site directed peptidyl aldehydes inhibit the cleavage of trans-

different substrate specificities. membrane substrate by a non-competitive mechanism, or in other

The TMD of the substrate was suggested to first interact with words, the inhibitor can bind to an enzyme-substrate complex [72].

an ‘intramembrane exosite’ of rhomboid, followed by a conforma- These lines of evidence collectively imply that the TMD of the sub-

tional change that might involve local unfolding of substrate TMD strate binds the enzyme first into a site that is spatially separated

and binding of the recognition motif region into rhomboid active from the active site, which fulfils the definition of an exosite [74],

site, resulting in scission [70] (Fig. 2A). This model is supported and then the recognition motif interacts with the active site leading

by several independent lines of evidence. First, separating the sub- to the proteolytic reaction, as proposed earlier [70].

strate TMD and recognition motif with a linker of increasing length This two-step mechanism implies that the Michaelis complex

gradually decreased the initial reaction rate [70]. Second, kinet- of rhomboid reaction (i.e. the enzyme-substrate complex that is in

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Fig. 2. Substrate recognition by rhomboid proteases.

A) Schematic model of events along the reaction co-ordinate of rhomboid catalysis. This model draws on the current consensus in the field assuming that the transmembrane

domain of the substrate makes first contact with rhomboid protease within lipid membrane (docking/interrogation complex). Only then does a linear sequence defining the

cleavage site (recognition motif) interact with rhomboid active site, forming the scission complex that aligns the polypeptide in the active site for the actual proteolytic

reaction to follow. The postulated intramembrane exosite is highlighted in brown, the recognition motif region in blue and catalytic dyad in red.

B) Structure of the complex of peptidyl chloromethylketone AcIATAcmk with GlpG (4qo2) was the first model of the acyl-enzyme and tetrahedral intermediate [71].

Chloromethylketones react covalently with both the catalytic Ser and His (Fig. 3D), forming a doubly bound tetrahedral uncharged species bearing similarity to the second

tetrahedral intermediate (forming during hydrolysis of the acyl-enzyme).

C) Structure of the complex of peptide aldehyde AcVRMAcho with GlpG (5f5b), the best available model of the second tetrahedral intermediate [72].

D) Model of the scission complex of rhomboid based on the crystal structure of a substrate peptide chloromethyketone complex (4qo2), molecular dynamics [71] and

manual docking of a model transmembrane helix of Providencia stuartii TatA. Aquiring experimental data on the structures of the transmembrane domain of the substrate

in the docking and scission complexes is the key missing element needed for the full understanding of the mechanism and specificity of rhomboid protease.

E) The spatial relationship of the proposed water retention site and S1 subsite in GlpG. The water retention site and the S1 subsite (both demarkated by dashed lines) are

part of a contiguous cavity inside GlpG that is displayed as a surface in the structure of the complex of peptidyl aldehyde AcVRMAcho with GlpG (5f5b). The cavity surface

is colored by hydrophobicity based on the YRB scheme [97] such that aliphatic hydrophobic carbons contribute yellow, negatively charged surfaces are red and positively

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thermodynamic equilibrium with free enzyme and substrate [75]) tein substrates and SDS-PAGE based readout [70,81–84]. While this

is actually the first encounter complex interacting via the trans- is an accurate and reproducible assay for the analysis of inhibi-

membrane regions of enzyme and substrate in which rhomboid tion [71,85], it is also low-throughput, relatively time-consuming,

active site is unoccupied [72]. Since the term Michaelis complex is and allows only an endpoint analysis of the reaction. An interest-

often used in a slightly different meaning, as the immediate pre- ing variation of this assay employs detection and quantitation of

scission complex of the enzyme and substrate poised for catalysis reaction products by mass spectrometry [86]. This assay however

[55,71,76], I herein instead adhere to the perhaps more explicitly requires an expensive instrument for detection and is not continu-

descriptive terms the ‘docking’ [77] or ‘interrogation’ [73] complex ous, which limits its practical use. Substrates that allow continuous

(Fig. 2A) for the first encounter complex, and ‘scission’ complex for assays and measurement of initial reaction rates using optical read-

the next stage complex where the substrate’s recognition motif is out are much more practical, but it has taken surprisingly long time

fully aligned in the active site of rhomboid (Fig. 2A). It is tempting to develop them.

to speculate that other rhomboid-superfamily proteins including A generic fluorogenic protease substrate, internally quenched

iRhoms and Derlins might share similar principles of recognition BODIPY-casein, has been reported to be cleaved by GlpG [54,82],

of their client proteins or substrates (see the article by Lemberg but the sites of cleavage are unknown, casein is a completely het-

and Adrain in this issue for more discussion). erologous substrate lacking a transmembrane domain, and thus

These two separable recognition features of rhomboid sub- does not mimic the interactions of a true rhomboid substrate.

strates operate in concert and are conformationally coupled. In While this might not be a problem for detecting active site directed

fact, the intimate connection between the lipid membrane and inhibitors, any inhibitors binding into the exosite region might not

structural dynamics of the transmembrane domain of substrate be detected. More native and advanced assays that enable mea-

may lie at the very heart of mechanism surement of use fluorogenic peptide or protein

[78]. Consistent with this idea, it has been demonstrated that substrates: a FRET derivative of the truncated Gurken substrate

membrane environment can influence the specificity of rhom- [85], a genetically encoded transmembrane FRET substrate based

boid protease: proteins that are normally non-substrates become on a CyPet-TatA-YPet recombinant fusion protein [87] or FITC

cleaved by rhomboid upon disturbing the membrane properties labelled TatA transmembrane peptide [73]. Each of these assays

chemically [79,80]. Similarly, perturbing the helicity of the TMD has its limitations: some work only for a specific rhomboid pro-

of a non-substrate by introducing prolines along the TMD turns it tease [85], others are usable only in liposomes [73], and yet others

into a substrate [80]. In summary, there is a gradual convergence of have a limited choice of fluorophores, are relatively large and have

opinions in the field which crystallizes in a model when two recog- a low FRET efficiency [87].

nition elements define rhomboid substrates [70] and the structural An alternative tool to substrate cleavage assays for detection of

dynamics of the substrate that determines their accessibility to proteases and their inhibitors are activity based probes [88]. A flu-

rhomboid is profoundly influenced by the membrane environment orescence polarization assay with activity based probes has been

[79]. reported for rhomboid proteases, and was used to identify new ␤-

The questions that remain to be answered to understand lactone type inhibitors [89] (discussed in more detail in Section 5.2).

intramembrane proteolysis by rhomboid proteases at atomic level Significant advantage of these phosphonofluoridate probes is that

concern the extent of the interaction surface between the enzyme they are relatively unselective, and might be usable for virtually any

and substrate, the identity of the hotspots or regions that determine rhomboid protease. On the other hand, fluorescence polarization

the binding energy, and the structure of the full substrate-enzyme is prone to detergent artifacts, and an active-site directed probe

complex. The first steps to the structural characterization of would most likely not detect an exosite-directed inhibitor. There

the substrate-enzyme complex were made recently. The X-ray is thus a need for a strategy to generate small fluorogenic trans-

structures of substrate-derived peptidyl chloromethylketone [71] membrane substrates that would capture all the relevant enzyme

(Fig. 2B) and peptidyl aldehyde inhibitors [72] (Fig. 2C) complexed substrate interactions, be more general or more specific for a par-

to GlpG mimic the acyl-enzyme and the second tetrahedral inter- ticular rhomboid protease as needed, be operating at red-shifted

mediate. These structures have revealed the mode of binding of wavelengths to be suitable for high-throughput screening of com-

the P4 to P1 residues into the corresponding subsites in the active pound libraries, and allow fast and continuous measurements in

site, and yielded a molecular model of the P4 to P3 fragment of detergent as well as liposomes.

the ‘scission’ complex [71] (Fig. 2D). This part of the substrate (the

recognition motif) binds into the active site as a parallel ␤-strand

with the L3 loop of GlpG stabilized by a number of hydrogen bonds 5. Rhomboid inhibitors

and van der Waals interactions (Fig. 2E and F). The P1 residue points

into a well-defined S1 subsite cavity, and the P4 residue interacts 5.1. The beginnings

with the L1 loop. The implications of these findings for inhibitor

design will be discussed in Section 5.2, and the implications for the The discovery that rhomboid is a had been aided

function of the L1 loop region in other rhomboid-family proteins by the sensitivity of the founding member Drosophila rhomboid-1

are discussed in more detail by Lemberg and Adrain in this issue. to serine protease inhibitors dichloroisocoumarin (DCI) and tosyl

phenylalanyl chloromethylketone (TPCK) [1]. The first structural

insights into the mechanism and specificity of rhomboid have been

4. Rhomboid activity assays gained from crystallographic studies of general serine protease

inhibitors diisopropylfluorophosphate [90], isocoumarins [90,91]

Inhibitor development requires robust activity assays for scor- and a phosphonofluoridate [92] (Fig. 3A and B). These studies have

ing of inhibitor efficiency and affinity. The first widely used in vivo revealed the oxyanion hole (Fig. 1C) and the likely S1 subsite of

or in vitro activity assays employed model transmembrane pro- rhomboid protease, but since the inhibitors were very dissim-

charged ones are in blue. The side-chains of residues proposed to stabilize the water molecules (red), the catalytic dyad and the P1 residue of the ligand are shown in green,

the rest of the peptide ligand is in grey. The S1 subsite has a hydrophobic surface, which is consistent with a strong preference for Ala in the P1 position by GlpG [71,91].

F) The structure of peptidyl aldehyde AcVRMAcho complexed to GlpG (5f5b) shows the non-prime subsites of GlpG. The surface of GlpG is colored by hydrophobicity (YRB

scheme [97]), the ligand is in green, and the subsites are approximated by dashed lines. The S4 subsite is formed by residues of the L1 loop.

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K. Strisovsky / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx 7

Fig. 3. The main classes and examples of rhomboid protease inhibitors. Reaction mechanisms with stereochemistry adjusted for rhomboid proteases (derived from the 3D

structures of inhibitor complexes with GlpG) are displayed as well as 3D structures of the inhibitor complexes and GlpG indicating the binding mode. Note that an isocoumarin

covalently binds to His150 (3zeb) instead of the more common His254 (2xow), and that a phosphonofluoridate (3ubb), isocoumarin (3zeb) and a ␤-lactam (3zmi) bind into

the prime side of rhomboid active site. A) phosphonofluoridates [90,92], B) isocoumarins [86,91], C) ␤-lactams and ␤-lactones [85,89,94], D) peptidyl chloromethylketones

[71], and E) peptidyl aldehydes [72]. The reversibility of binding of peptidyl aldehydes is denoted by a reverse arrow.

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8 K. Strisovsky / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

ilar from a peptide substrate, they were not more informative boid, or in fact, any intramembrane protease [71] (discussed also

about rhomboid mechanism and specificity. All of these inhibitors in Section 3.2). Recent work shows that the active site of rhomboid

are based on a very reactive electrophile, hence likely to inhibit can accommodate about 6 residues (P4 to P2 ), and displays distinct

other serine proteases, and they should definitely not be used as sequence preferences at several positions [70,71] Most observa-

rhomboid-specific inhibitors in cell biological studies (which DCI tions made from the structures of peptidyl chloromethylketones

sometimes is, e.g. [35,37]). The lack of selectivity is a disadvantage [71] have been independently confirmed by recent structures of

for cell biological use, but it is a distinct advantage for the genera- GlpG complexed to peptidyl aldehydes [72] (Fig. 3E), and these

tion of activity-based probes (ABPs) useful for a general rhomboid two studies together pave the way for a next generation of rhom-

assay [45,86,89,93] (discussed in Section 4). boid inhibitors based on an oligopeptide (or its mimic) coupled

Improvements of rhomboid activity assays enabled screening to a suitable electrophilic warhead. Their rational design will be

of compound libraries for rhomboid inhibitors. A high-throughput undoubtedly guided by structure of their complexes with GlpG and

assay of about 60,000-compound library with a fluorogenic FRET may be aided by an improved understanding of rhomboid mecha-

substrate and AarA revealed monocyclic ␤-lactams as new rhom- nism (discussed in Section 3.1).

boid inhibitors [85] (Fig. 3C). These compounds covalently bind the The crystal structures of the complexes of GlpG with the pep-

catalytic serine of GlpG, show time-dependence of inhibition, slow tide inhibitors have spurred a speculative hypothesis that rests in

reversibility, and their potency in vitro is low micromolar or sub- the fact that GlpG shows a strict preference for small amino acids

micromolar on Providencia stuartii AarA and E. coli GlpG (under a in the P1 position (such as Ala), but the S1 subsite is a larger cavity

30 min pre-incubation with the enzyme) [85]. They display some into which larger residues could theoretically be accommodated.

selectivity between GlpG and AarA over chymotrypsin [85], but Interestingly, the ‘deeper’ parts of the S1 subsite coincide with the

their activity in vivo is limited, and even at high micromolar con- previously postulated water retention site [63] (discussed in Sec-

centrations ␤-lactams do not inhibit the endogenous E. coli GlpG tion 3.1) (Fig. 2E). It is therefore possible that small amino acids

fully [85]. This could reflect the fact that the complexes of the in the P1 position are preferred by rhomboids because larger ones

monocyclic ␤-lactams with GlpG slowly hydrolyse in a way that might interfere with the water transfer from the retention site to

leads to the inactivation of the inhibitor, because the ␤-lactam ring the catalytic dyad during the catalytic cycle [71]. If this mechanism

is not reformed spontaneously in solution. ␤-lactams are there- is confirmed, modification of the P1 residue of the inhibitor such

fore in fact slowly turned over by rhomboid and act rather as that it allows binding into the S1 subsite but restricts or blocks

‘failed-suicide substrates’. Crystallographic analyses of complexes water transfer from the water retention site would constitute an

of these compounds with GlpG show that they occupy the prime entirely new mechanism to increase the selectivity of rhomboid

side of rhomboid active site, possibly the S2 subsite [94] (Fig. 3C). inhibitors.

Structure-activity relationship analysis has indicated how mono- All the so far discussed rhomboid inhibitors covalently bind

cyclic ␤-lactams could be modified to enhance their selectivity [85], one or both residues of the catalytic dyad via a reactive elec-

but surprisingly these compounds have not been developed further trophilic group. This strategy in principle may limit selectivity,

so far. because many other serine proteases (or in general)

A lot of inhibitor development effort has since then focused employing similar catalytic chemistry might be also affected by

on isocoumarins. Screening of a library of electrophiles includ- these compounds. None of the so far developed rhomboid inhibitors

ing isocoumarins, phosphonates and sulphonyl fluorides using a have been tested for selectivity beyond measuring their effect on

MALDI mass-spectrometry based assay yielded several new iso- common digestive proteases chymotrypsin or trypsin [85,86]. In

coumarins that were developed into activity-based probes [86]. In general, a more reactive electrophilic ‘warhead’ leads to a more

an effort to identify new electrophile chemistries that would be potent inhibitor, but with a lower selectivity. In practice, selectivity

active as rhomboid inhibitors, a small library of 85 compounds can be improved by target-specific modification of the inhibitor. A

of reactive electrophiles including isocoumarins, phosphonates, big challenge for future studies will thus be controlling and improv-

phosphoramidates, ␤-sultams, ␤-lactones, epoxides and thiiranes, ing inhibitor selectivity while maintaining potency. One strategy

has been screened using a novel activity assay based on ABP flu- could be to use a less reactive electrophilic warhead while adding

orescence polarization [89]. The ␤-lactones have been thereby specificity-determining moieties to ensure tight binding. Exploit-

identified as a new class of rhomboid inhibitors (Fig. 3C). They are ing the knowledge of rhomboid mechanism and subsite preferences

structurally similar to the ␤-lactams, are relatively weak inhibitors [70–73], sequence-optimized oligopeptides or their mimics could

with apparent IC50 in high micromolar range against rhomboid, be explored that will be coupled to a variety of electrophiles that

but ␤-lactone inhibitors and ABPs at least show some selectivity have been successfully used in protease inhibitors and are even

among rhomboids [45]. The inhibition properties of isocoumarins, clinically used, such as peptidyl boronates, nitriles, or various

␤-lactams and ␤-lactones can probably be further improved, but activated ketones [reviewed in [53,95]]. In addition, since some

insufficient selectivity will probably be a persistent problem with isocoumarins and ␤-lactams bind into the prime side of rhomboid

isocoumarins due to their high reactivity and irreversible chem- active site (Fig. 3B and C), designing inhibitors that would occupy

istry, generally undesired for pharmacological use. The ␤-lactams both the prime and non-prime sides of the active site could increase

seem the best candidates for further development, but given their their affinity and selectivity.

limited stability and in vivo activity discussed above, there is defi- If we think beyond active-site directed inhibitors, we realize

nitely a clear scope to explore the chemical space for new, potent that binding of substrate TMD at an intramembrane exosite [70,71]

and selective rhomboid inhibitors. (also called an ‘interrogation site’ [72,73]) of GlpG is the first step

in catalysis, which suggests that exosite-directed non-covalent

5.2. Structure and mechanism based design and future directions inhibitors might be an alternative. Indeed, helical have

been designed that efficiently inhibit ␥-secretase [96], and their

The desire to understand the structural basis of rhomboid mechanism of action likely involves binding into an initial ‘docking

specificity and sequence preferences [70] has led to the design site’, supporting the general feasibility of this approach. It is uncer-

of substrate-derived peptidyl chloromethylketone inhibitors [71] tain whether such compounds could in principle achieve selectivity

(Fig. 3D). These compounds interacted with GlpG in a substrate- for rhomboids, because recent work suggests that binding at the

like manner, and their co-crystal structures with GlpG offered the rhomboid docking site/exosite might be relatively weak and not

first structural insight into the mode of substrate binding to rhom- very selective [73]. Nonetheless, this area is certainly worth explor-

Please cite this article in press as: K. Strisovsky, Rhomboid protease inhibitors: Emerging tools and future therapeutics, Semin Cell Dev

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ing further, and development of more efficient activity assays that [14] L.A. Baxt, E. Rastew, R. Bracha, D. Mirelman, U. Singh, Downregulation of an

entamoeba histolytica rhomboid protease reveals roles in regulating parasite

could detect binding to the intramembrane exosite while being

adhesion and phagocytosis, Eukaryot. Cell 9 (8) (2010) 1283–1293.

suitable for high-throughput screening would greatly facilitate this

[15] L.A. Baxt, R.P. Baker, U. Singh, S. Urban, An Entamoeba histolytica rhomboid

effort. protease with atypical specificity cleaves a surface lectin involved in

phagocytosis and immune evasion, Genes Dev. 22 (12) (2008) 1636–1646.

[16] R.P. Baker, R. Wijetilaka, S. Urban, Two Plasmodium rhomboid proteases

6. Concluding remarks and perspectives

preferentially cleave different adhesins implicated in all invasive stages of

malaria, PLoS Pathog. 2 (10) (2006) e113.

[17] R.A. O’Donnell, F. Hackett, S.A. Howell, M. Treeck, N. Struck, Z. Krnajski, C.

Rhomboid intramembrane proteases are becoming increas-

Withers-Martinez, T.W. Gilberger, M.J. Blackman, Intramembrane proteolysis

ingly attractive in disease contexts, but the development of their

mediates shedding of a key adhesin during erythrocyte invasion by the

inhibitors has lagged behind. It will be important to develop selec- malaria parasite, J. Cell Biol. 174 (7) (2006) 1023–1033.

[18] J.W. Lin, P. Meireles, M. Prudencio, S. Engelmann, T. Annoura, M. Sajid, S.

tive and potent inhibitors of rhomboids both for exploration and

Chevalley-Maurel, J. Ramesar, C. Nahar, C.M. Avramut, A.J. Koster, K.

validation of their physiological roles and for prospective drug

Matuschewski, A.P. Waters, C.J. Janse, G.R. Mair, S.M. Khan, Loss-of-function

development. These efforts will be aided by improved activity analyses defines vital and redundant functions of the Plasmodium rhomboid

assays and deeper mechanistic and structural understanding of protease family, Mol. Microbiol. 88 (2) (2013) 318–338.

[19] Y. Vaknin, F. Hillmann, R. Iannitti, N. Ben Baruch, H. Sandovsky-Losica, Y.

rhomboid proteases. The near future could bring further develop-

Shadkchan, L. Romani, A. Brakhage, O. Kniemeyer, N. Osherov, Identification

ment of existing covalently binding scaffolds such as the ␤-lactams,

and, Characterization of a novel aspergillus fumigatus rhomboid family

discovery of new covalent active site directed inhibitors as well as putative protease, RbdA, involved in hypoxia sensing and virulence, Infect.

Immun. 84 (6) (2016) 1866–1878.

novel, exosite-binding or allosteric inhibitors.

[20] L.G. Stevenson, K. Strisovsky, K.M. Clemmer, S. Bhatt, M. Freeman, P.N. Rather,

Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by

Acknowledgements activating TatA of the twin-arginine , Proc. Natl. Acad. Sci. U. S. A.

104 (3) (2007) 1003–1008.

[21] L.R. Mesak, F.M. Mesak, M.K. Dahl, Expression of a novel gene, gluP, is

I acknowledge support from EMBO (Installation Grant no. 2329),

essential for normal Bacillus subtilis cell division and contributes to glucose

Ministry of Education, Youth and Sports of the Czech Republic export, BMC Microbiol. 4 (2004) 13.

[22] J. Parente, A. Casabuono, M.C. Ferrari, R.A. Paggi, R.E. De Castro, A.S. Couto, M.I.

(projects no. LK11206 and LO1302), Marie Curie Career Integration

Gimenez, A rhomboid protease gene deletion affects a novel oligosaccharide

Grant (project no. 304154), and the National Subvention for Devel-

N-linked to the S-layer glycoprotein of Haloferax volcanii, J. Biol. Chem. 289

opment of Research Organisations (RVO: 61388963) to the Institute (16) (2014) 11304–11317.

[23] D.P. Kateete, F.A. Katabazi, A. Okeng, M. Okee, C. Musinguzi, B.B. Asiimwe, S.

of Organic Chemistry and Biochemistry. I thank Pavel Majer and

Kyobe, J. Asiimwe, W.H. Boom, M.L. Joloba, Rhomboids of mycobacteria:

Martin Lepsíkˇ for critical reading and feedback on the manuscript.

characterization using an aarA mutant of providencia stuartii and gene

deletion in mycobacterium smegmatis, PLoS One 7 (9) (2012) e45741.

References [24] L.N. Kinch, N.V. Grishin, perspective on rhomboid

intramembrane protease evolution and function, Biochim. Biophys. Acta 1828

(12) (2013) 2937–2943.

[1] S. Urban, J.R. Lee, M. Freeman, Drosophila rhomboid-1 defines a family of

[25] C.P. Blobel, ADAMs: key components in EGFR signalling and development,

putative intramembrane serine proteases, Cell 107 (2) (2001) 173–182.

Nat. Rev. Mol. Cell Biol. 6 (1) (2005) 32–43.

[2] S. Urban, J.R. Lee, M. Freeman, A family of Rhomboid intramembrane

[26] K. Saftig, The a disintegrin and metalloproteases ADAM10 and ADAM17:

proteases activates all Drosophila membrane-tethered EGF ligands, EMBO J.

novel drug targets with therapeutic potential? Eur. J. Cell Biol. 90 (6–7) (2011)

21 (16) (2002) 4277–4286.

527–535.

[3] E.V. Koonin, K.S. Makarova, I.B. Rogozin, L. Davidovic, M.C. Letellier, L.

[27] M.A. Brooke, S.L. Etheridge, N. Kaplan, C. Simpson, E.A. O’Toole, A.

Pellegrini, The rhomboids: a nearly ubiquitous family of intramembrane

Ishida-Yamamoto, O. Marches, S. Getsios, D.P. Kelsell, iRHOM2-dependent

serine proteases that probably evolved by multiple ancient horizontal gene

regulation of ADAM17 in cutaneous disease and epidermal barrier function,

transfers, Genome Biol. 4 (3) (2003) R19.

Hum. Mol. Genet. 23 (15) (2014) 4064–4076.

[4] M. Freeman, The rhomboid-like superfamily: molecular mechanisms and

[28] V. Hosur, K.R. Johnson, L.M. Burzenski, T.M. Stearns, R.S. Maser, L.D. Shultz,

biological roles, Annu. Rev. Cell Dev. Biol. 30 (2014) 235–254.

Rhbdf2 mutations increase its protein stability and drive EGFR

[5] C. Adrain, M. Zettl, Y. Christova, N. Taylor, M. Freeman, Tumor necrosis factor

hyperactivation through enhanced secretion of amphiregulin, Proc. Natl.

signaling requires iRhom2 to promote trafficking and activation of TACE,

Acad. Sci. U. S. A. 111 (21) (2014) E2200–9.

Science 335 (6065) (2012) 225–228.

[29] Y. Christova, C. Adrain, P. Bambrough, A. Ibrahim, M. Freeman, Mammalian

[6] D.R. McIlwain, P.A. Lang, T. Maretzky, K. Hamada, K. Ohishi, S.K. Maney, T.

iRhoms have distinct physiological functions including an essential role in

Berger, A. Murthy, G. Duncan, H.C. Xu, K.S. Lang, D. Haussinger, A. Wakeham,

TACE regulation, EMBO Rep. 14 (10) (2013) 884–890.

A. Itie-Youten, R. Khokha, P.S. Ohashi, C.P. Blobel, T.W. Mak, iRhom2

[30] C. Adrain, K. Strisovsky, M. Zettl, L. Hu, M.K. Lemberg, M. Freeman,

regulation of TACE controls TNF-mediated protection against Listeria and

Mammalian EGF receptor activation by the rhomboid protease RHBDL2,

responses to LPS, Science 335 (6065) (2012) 229–232.

EMBO Rep. 12 (5) (2011) 421–427.

[7] P.D. Issuree, T. Maretzky, D.R. McIlwain, S. Monette, X. Qing, P.A. Lang, S.L.

[31] H.J. Liao, G. Carpenter, Regulated intramembrane cleavage of the EGF

Swendeman, K.H. Park-Min, N. Binder, G.D. Kalliolias, A. Yarilina, K. Horiuchi,

receptor, Traffic 13 (8) (2012) 1106–1112.

L.B. Ivashkiv, T.W. Mak, J.E. Salmon, C.P. Blobel, iRHOM2 is a critical

[32] W. Song, W. Liu, H. Zhao, S. Li, X. Guan, J. Ying, Y. Zhang, F. Miao, M. Zhang, X.

pathogenic mediator of inflammatory arthritis, J. Clin. Invest. 123 (2) (2013)

Ren, X. Li, F. Wu, Y. Zhao, Y. Tian, W. Wu, J. Fu, J. Liang, W. Wu, C. Liu, J. Yu, S.

928–932.

Zong, S. Miao, X. Zhang, L. Wang, Rhomboid domain containing 1 promotes

[8] X. Li, T. Maretzky, G. Weskamp, S. Monette, X. Qing, P.D. Issuree, H.C.

colorectal cancer growth through activation of the EGFR signalling pathway,

Crawford, D.R. McIlwain, T.W. Mak, J.E. Salmon, C.P. Blobel, iRhoms 1 and 2

Nat. Commun. 6 (2015) 8022.

are essential upstream regulators of ADAM17-dependent EGFR signaling,

[33] M.K. Lemberg, M. Freeman, Functional and evolutionary implications of

Proc. Natl. Acad. Sci. U. S. A. 112 (19) (2015) 6080–6085.

enhanced genomic analysis of rhomboid intramembrane proteases, Genome

[9] S.K. Maney, D.R. McIlwain, R. Polz, A.A. Pandyra, B. Sundaram, D. Wolff, K.

Res. 17 (11) (2007) 1634–1646.

Ohishi, T. Maretzky, M.A. Brooke, A. Evers, A.A. Vasudevan, N. Aghaeepour, J.

[34] O. Lohi, S. Urban, M. Freeman, Diverse substrate recognition mechanisms for

Scheller, C. Munk, D. Haussinger, T.W. Mak, G.P. Nolan, D.P. Kelsell, C.P. Blobel,

rhomboids; is cleaved by Mammalian rhomboids, Curr. Biol.

K.S. Lang, P.A. Lang, Deletions in the cytoplasmic domain of iRhom1 and

14 (3) (2004) 236–241.

iRhom2 promote shedding of the TNF receptor by the protease ADAM17, Sci.

[35] T.L. Cheng, Y.T. Wu, H.Y. Lin, F.C. Hsu, S.K. Liu, B.I. Chang, W.S. Chen, C.H. Lai,

Signal. 8 (401) (2015) ra109.

G.Y. Shi, H.L. Wu, Functions of rhomboid family protease RHBDL2 and

[10] O.M. Siggs, A. Grieve, H. Xu, P. Bambrough, Y. Christova, M. Freeman, Genetic

thrombomodulin in wound healing, J. Invest. Dermatol. 131 (12) (2011)

interaction implicates iRhom2 in the regulation of EGF receptor signalling in

2486–2494.

mice, Biol. Open 3 (12) (2014) 1151–1157.

[36] P.J. Noy, R.K. Swain, K. Khan, P. Lodhia, R. Bicknell, Sprouting angiogenesis is

[11] J.M. Santos, A. Graindorge, D. Soldati-Favre, New insights into parasite

regulated by shedding of the C-type lectin family 14, member A (CLEC14A)

rhomboid proteases, Mol. Biochem. Parasitol. 182 (1-2) (2012) 27–36.

ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2), FASEB J. 30 (6)

[12] L.D. Sibley, The roles of intramembrane proteases in protozoan parasites,

(2016) 2311–2323.

Biochim. Biophys. Acta 1828 (12) (2013) 2908–2915.

[37] T.L. Cheng, C.H. Lai, S.J. Jiang, J.H. Hung, S.K. Liu, B.I. Chang, G.Y. Shi, H.L. Wu,

[13] A.M. Riestra, S. Gandhi, M.J. Sweredoski, A. Moradian, S. Hess, S. Urban, P.J.

RHBDL2 is a critical membrane protease for anoikis resistance in human

Johnson, A trichomonas vaginalis rhomboid protease and its substrate

malignant epithelial cells, Sci. World J. 2014 (2014) 902987.

modulate parasite attachment and cytolysis of host cells, PLoS Pathog. 11 (12)

(2015) e1005294.

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

G Model

YSCDB-2122; No. of Pages 11 ARTICLE IN PRESS

10 K. Strisovsky / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

[38] R. Canzoneri, E. Lacunza, M. Isla Larrain, M.V. Croce, M.C. Abba, Rhomboid [65] P.K. Madala, J.D. Tyndall, T. Nall, D.P. Fairlie, Update 1 of: proteases universally

family gene expression profiling in breast normal tissue and tumor samples, recognize beta strands in their active sites, Chem. Rev. 110 (6) (2010)

Tumour Biol. 35 (2) (2014) 1451–1458. PR1–31.

[39] L. Fleig, N. Bergbold, P. Sahasrabudhe, B. Geiger, L. Kaltak, M.K. Lemberg, [66] J.D. Tyndall, T. Nall, D.P. Fairlie, Proteases universally recognize beta strands in

Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of their active sites, Chem. Rev. 105 (3) (2005) 973–999.

membrane proteins, Mol. Cell 47 (4) (2012) 558–569. [67] S.C. Li, C.M. Deber, A measure of helical propensity for amino acids in

[40] L. Wunderle, J.D. Knopf, N. Kuhnle, A. Morle, B. Hehn, C. Adrain, K. Strisovsky, membrane environments, Nat. Struct. Biol. 1 (6) (1994) 368–373.

M. Freeman, M.K. Lemberg, Rhomboid intramembrane protease RHBDL4 [68] S. Urban, M. Freeman, Substrate specificity of rhomboid intramembrane

triggers ER-export and non-canonical secretion of membrane-anchored proteases is governed by helix-breaking residues in the substrate

TGFalpha, Sci. Rep. 6 (2016) 27342. transmembrane domain, Mol. Cell 11 (6) (2003) 1425–1434.

[41] J.C. Pascall, K.D. Brown, Characterization of a mammalian cDNA encoding a [69] Y. Akiyama, S. Maegawa, Sequence features of substrates required for

protein with high sequence similarity to the Drosophila regulatory protein cleavage by GlpG, an rhomboid protease, Mol. Microbiol. 64

Rhomboid, FEBS Lett. 429 (3) (1998) 337–340. (4) (2007) 1028–1037.

[42] J. Jaszai, M. Brand, Cloning and expression of Ventrhoid, a novel vertebrate [70] K. Strisovsky, H.J. Sharpe, M. Freeman, Sequence-specific intramembrane

homologue of the Drosophila EGF pathway gene rhomboid, Mech. Dev. 113 proteolysis: identification of a recognition motif in rhomboid substrates, Mol.

(1) (2002) 73–77. Cell 36 (6) (2009) 1048–1059.

[43] A. Petri, J. Ahnfelt-Ronne, K.S. Frederiksen, D.G. Edwards, D. Madsen, P. Serup, [71] S. Zoll, S. Stanchev, J. Began, J. Skerle, M. Lepsik, L. Peclinovska, P. Majer, K.

J. Fleckner, R.S. Heller, The effect of neurogenin3 deficiency on pancreatic Strisovsky, Substrate binding and specificity of rhomboid intramembrane

gene expression in embryonic mice, J. Mol. Endocrinol. 37 (2) (2006) 301–316. protease revealed by substrate-peptide complex structures, EMBO J. 33 (20)

[44] A. Kumar, J.R. Gibbs, A. Beilina, A. Dillman, R. Kumaran, D. Trabzuni, M. Ryten, (2014) 2408–2421.

R. Walker, C. Smith, B.J. Traynor, J. Hardy, A.B. Singleton, M.R. Cookson, [72] S. Cho, S.W. Dickey, S. Urban, Crystal structures and inhibition kinetics reveal

Age-associated changes in gene expression in human brain and isolated a two-stage catalytic mechanism with drug design implications for rhomboid

neurons, Neurobiol. Aging 34 (4) (2013) 1199–1209. proteolysis, Mol. Cell 61 (3) (2016) 329–340.

[45] E.V. Wolf, A. Zeissler, S.H. Verhelst, Inhibitor fingerprinting of rhomboid [73] S.W. Dickey, R.P. Baker, S. Cho, S. Urban, Proteolysis inside the membrane is a

proteases by activity-Based protein profiling reveals inhibitor selectivity and rate-governed reaction not driven by substrate affinity, Cell 155 (6) (2013)

rhomboid autoprocessing, ACS Chem. Biol. 10 (10) (2015) 2325–2333. 1270–1281.

[46] S. Cipolat, T. Rudka, D. Hartmann, V. Costa, L. Serneels, K. Craessaerts, K. [74] C.M. Overall, Molecular determinants of substrate

Metzger, C. Frezza, W. Annaert, L. D’Adamio, C. Derks, T. Dejaegere, L. specificity: matrix metalloproteinase substrate binding domains, modules,

Pellegrini, R. D’Hooge, L. Scorrano, B. De Strooper, Mitochondrial rhomboid and exosites, Mol. Biotechnol. 22 (1) (2002) 51–86.

PARL regulates cytochrome c release during apoptosis via OPA1-dependent [75] R.A. Copeland, Enzyme Reaction Mechanisms, Evaluation of Enzyme

cristae remodeling, Cell 126 (1) (2006) 163–175. Inhibitors in Drug Discovery, John Wiley & Sons, Inc., 2013, pp. 25–55.

[47] E.Y. Chan, G.A. McQuibban, The mitochondrial rhomboid protease: its rise [76] E.S. Radisky, J.M. Lee, C.J. Lu, D.E. Koshland Jr., Insights into the serine protease

from obscurity to the pinnacle of disease-relevant genes, Biochim. Biophys. mechanism from atomic resolution structures of trypsin reaction

Acta 1828 (12) (2013) 2916–2925. intermediates, Proc. Natl. Acad. Sci. U. S. A. 103 (18) (2006)

[48] C. Meissner, H. Lorenz, B. Hehn, M.K. Lemberg, Intramembrane protease PARL 6835–6840.

defines a negative regulator of PINK1- and PARK2/Parkin-dependent [77] K. Strisovsky, Why cells need intramembrane proteases – a mechanistic

mitophagy, Autophagy 11 (9) (2015) 1484–1498. perspective, FEBS J. 283 (10) (2016) 1837–1845.

[49] C.T. Chu, A pivotal role for PINK1 and autophagy in mitochondrial quality [78] D. Langosch, C. Scharnagl, H. Steiner, M.K. Lemberg, Understanding

control: implications for Parkinson disease, Hum. Mol. Genet. 19 (R1) (2010) intramembrane proteolysis: from protein dynamics to reaction kinetics,

R28–37. Trends Biochem. Sci. 40 (6) (2015) 318–327.

[50] N. Exner, A.K. Lutz, C. Haass, K.F. Winklhofer, Mitochondrial dysfunction in [79] S. Urban, S.M. Moin, A subset of membrane-altering agents and

Parkinson’s disease: molecular mechanisms and pathophysiological gamma-secretase modulators provoke nonsubstrate cleavage by rhomboid

consequences, EMBO J. 31 (14) (2012) 3038–3062. proteases, Cell Rep. 8 (5) (2014) 1241–1247.

[51] P.M. Abou-Sleiman, M.M. Muqit, N.W. Wood, Expanding insights of [80] S.M. Moin, S. Urban, Membrane immersion allows rhomboid proteases to

mitochondrial dysfunction in Parkinson’s disease, Nat. Rev. Neurosci. 7 (3) achieve specificity by reading transmembrane segment dynamics, eLife 1

(2006) 207–219. (2012) e00173.

[52] J. Szendroedi, E. Phielix, M. Roden, The role of mitochondria in insulin [81] S. Urban, D. Schlieper, M. Freeman, Conservation of intramembrane

resistance and type 2 diabetes mellitus, Nat. Rev. Endocrinol. 8 (2) (2012) proteolytic activity and substrate specificity in prokaryotic and eukaryotic

92–103. rhomboids, Curr. Biol. 12 (17) (2002) 1507–1512.

[53] L. Hedstrom, Serine protease mechanism and specificity, Chem. Rev. 102 (12) [82] S. Maegawa, K. Ito, Y. Akiyama, Proteolytic action of GlpG, a rhomboid

(2002) 4501–4524. protease in the Escherichia coli cytoplasmic membrane, Biochemistry 44 (41)

[54] Y. Wang, Y. Zhang, Y. Ha, Crystal structure of a rhomboid family (2005) 13543–13552.

intramembrane protease, Nature 444 (7116) (2006) 179–180. [83] M.K. Lemberg, J. Menendez, A. Misik, M. Garcia, C.M. Koth, M. Freeman,

[55] N. Uritsky, M. Shokhen, A. Albeck, The catalytic machinery of rhomboid Mechanism of intramembrane proteolysis investigated with purified

proteases: combined MD and QM simulations, J. Chem. Theory Comput. 8 (11) rhomboid proteases, EMBO J. 24 (3) (2005) 464–472.

(2012) 4663–4671. [84] S. Urban, M.S. Wolfe, Reconstitution of intramembrane proteolysis in vitro

[56] N. Uritsky, M. Shokhen, A. Albeck, Stepwise versus concerted mechanisms in reveals that pure rhomboid is sufficient for catalysis and specificity, Proc.

general-Base catalysis by serine proteases, Angew. Chem. Int. Ed. Engl. 55 (5) Natl. Acad. Sci. U. S. A. 102 (6) (2005) 1883–1888.

(2016) 1680–1684. [85] O.A. Pierrat, K. Strisovsky, Y. Christova, J. Large, K. Ansell, N. Bouloc, E.

[57] Z. Wu, N. Yan, L. Feng, A. Oberstein, H. Yan, R.P. Baker, L. Gu, P.D. Jeffrey, S. Smiljanic, M. Freeman, Monocyclic beta-lactams are selective,

Urban, Y. Shi, Structural analysis of a rhomboid family intramembrane mechanism-based inhibitors of rhomboid intramembrane proteases, ACS

protease reveals a gating mechanism for substrate entry, Nat. Struct. Mol. Chem. Biol. 6 (4) (2011) 325–335.

Biol. 13 (12) (2006) 1084–1091. [86] O. Vosyka, K.R. Vinothkumar, E.V. Wolf, A.J. Brouwer, R.M. Liskamp, S.H.

[58] M.J. Lemieux, S.J. Fischer, M.M. Cherney, K.S. Bateman, M.N. James, The crystal Verhelst, Activity-based probes for rhomboid proteases discovered in a mass

structure of the rhomboid peptidase from Haemophilus influenzae provides spectrometry-based assay, Proc. Natl. Acad. Sci. U. S. A. 110 (7) (2013)

insight into intramembrane proteolysis, Proc. Natl. Acad. Sci. USA 104 (3) 2472–2477.

(2007) 750–754. [87] E. Arutyunova, P. Panwar, P.M. Skiba, N. Gale, M.W. Mak, M.J. Lemieux,

[59] M.N.G. James, Convergence of active-center geometries among the of rhomboid intramembrane proteolysis, EMBO J. 33 (17)

proteolytic-enzymes, in: J.S. Bond, A.J. Barrett (Eds.), Proteolysis and Protein (2014) 1869–1881.

Turnover, Portland Press Ltd., London, 1993, pp. 1–8. [88] D.A. Bachovchin, S.J. Brown, H. Rosen, B.F. Cravatt, Identification of selective

[60] DD.a. Ekici, M. Paetzel, R.E. Dalbey, Unconventional serine proteases: inhibitors of uncharacterized enzymes by high-throughput screening with

variations on the catalytic Ser/His/Asp triad configuration, Protein Sci. 17 (12) fluorescent activity-based probes, Nat. Biotechnol. 27 (4) (2009)

(2008) 2023–2037. 387–394.

[61] M. Paetzel, R.E. Dalbey, N.C. Strynadka, Crystal structure of a bacterial signal [89] E.V. Wolf, A. Zeissler, O. Vosyka, E. Zeiler, S. Sieber, S.H. Verhelst, A new class

peptidase in complex with a beta-lactam inhibitor, Nature 396 (6707) (1998) of rhomboid protease inhibitors discovered by activity-based fluorescence

186–190. polarization, PLoS One 8 (8) (2013) e72307.

[62] R. Henderson, Structure of crystalline alpha-chymotrypsin. IV. The structure [90] Y. Xue, Y. Ha, Catalytic mechanism of rhomboid protease GlpG probed by

of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, J. Biol. Chem.

mechanism of the enzyme, J. Mol. Biol. 54 (2) (1970) 341–354. 287 (5) (2012) 3099–3107.

[63] Y. Zhou, S.M. Moin, S. Urban, Y. Zhang, An internal water-retention site in the [91] K.R. Vinothkumar, K. Strisovsky, A. Andreeva, Y. Christova, S. Verhelst, M.

rhomboid intramembrane protease GlpG ensures catalytic efficiency, Freeman, The structural basis for catalysis and substrate specificity of a

Structure 20 (7) (2012) 1255–1263. rhomboid protease, EMBO J. 29 (22) (2010) 3797–3809.

[64] J.C. Timmer, W. Zhu, C. Pop, T. Regan, S.J. Snipas, A.M. Eroshkin, S.J. Riedl, G.S. [92] Y. Xue, S. Chowdhury, X. Liu, Y. Akiyama, J. Ellman, Y. Ha, Conformational

Salvesen, Structural and kinetic determinants of protease substrates, Nat. change in rhomboid protease GlpG induced by inhibitor binding to its S’

Struct. Mol. Biol. 16 (10) (2009) 1101–1108. subsites, Biochemistry 51 (18) (2012) 3723–3731.

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

G Model

YSCDB-2122; No. of Pages 11 ARTICLE IN PRESS

K. Strisovsky / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx 11

[93] E.V. Wolf, M. Seybold, R. Hadravova, K. Strisovsky, S.H. Verhelst, [96] C. Das, O. Berezovska, T.S. Diehl, C. Genet, I. Buldyrev, J.Y. Tsai, B.T. Hyman,

Activity-based protein profiling of rhomboid proteases in liposomes, M.S. Wolfe, Designed helical peptides inhibit an intramembrane protease, J.

ChemBioChem 16 (11) (2015) 1616–1621. Am. Chem. Soc. 125 (39) (2003) 11794–11795.

[94] K.R. Vinothkumar, O.A. Pierrat, J.M. Large, M. Freeman, Structure of rhomboid [97] D. Hagemans, I.A. van Belzen, T. Moran Luengo, S.G. Rudiger, A script to

protease in complex with beta-Lactam inhibitors defines the S2’ cavity, highlight hydrophobicity and charge on protein surfaces, Front. Mol. Biosci. 2

Structure 21 (6) (2013) 1051–1058. (2015) 56.

[95] B. Walker, J.F. Lynas, Strategies for the inhibition of serine proteases, Cell. Mol.

Life Sci. 58 (4) (2001) 596–624.

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