Antibiotic Targeting of the Bacterial Secretory Pathway☆

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Biochimica et Biophysica Acta 1843 (2014) 1762–1783

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Review Antibiotic targeting of the bacterial secretory pathway☆

Smitha Rao C.V. a, Evelien De Waelheyns a, Anastassios Economou a,b,c,JozefAnnéa,⁎

a Laboratory of Molecular Bacteriology, Rega Institute, Department of Microbiology and Immunology, KU Leuven, O&N1, 6th ﬂoor, Herestraat 49, P.O. Box 1037, B-3000 Leuven, Belgium b Institute of Molecular Biology and Biotechnology, FORTH, University of Crete, P.O. Box 1385, GR-711 10 Iraklio, Crete, Greece c Department of Biology, University of Crete, P.O. Box 1385, GR-71110 Iraklio, Crete, Greece

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Article history: Finding new, effective antibiotics is a challenging research area driven by novel approaches required to tackle un- Received 31 October 2013 conventional targets. In this review we focus on the bacterial protein secretion pathway as a target for eliminating Received in revised form 27 January 2014 or disarming pathogens. We discuss the latest developments in targeting the Sec-pathway for novel antibiotics Accepted 6 February 2014 focusing on two key components: SecA, the ATP-driven motor protein responsible for driving preproteins across Available online 15 February 2014 the cytoplasmic membrane and the Type I signal peptidase that is responsible for the removal of the signal peptide allowing the release of the mature protein from the membrane. We take a bird's-eye view of other potential tar- Keywords: Antibiotic target gets in the Sec-pathway as well as other Sec-dependent or Sec-independent protein secretion pathways as targets Protein secretion inhibitor for the development of novel antibiotics. This article is part of a Special Issue entitled: Protein trafﬁcking and se- Signal peptidase cretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. SecA © 2014 Elsevier B.V. All rights reserved. Antibiotic resistance

1. Introduction to a particular antibiotic that often results in resistance to other antibiotics, usually from a similar chemical class, to which the bacteria may 1.1. Antibacterial resistance and the dire need for new antibiotics not have been exposed). The past thirty years have seen only two truly novel classes of antibiotics entering the market: the oxalidiones (linezo- Due to their short life cycle and ability to adapt quickly to changes in lid) and cyclic lipopeptides (daptomycin), for which resistant strains the environment, pathogenic bacteria continue to persist by gradually have already emerged [3]. The problem of antibiotic resistance began es- overcoming the effect of drugs used to eradicate them. This natural calating since the 1980s with a progressive increase in the number of in- phenomenon of drug resistance is often hastened by human practices fections due to methicillin-resistant Staphylococcus aureus (MRSA), due to inappropriate use of antibiotics. Currently, more than 70% of vancomycin-resistant Enterococcus and fluoroquinolone resistant Pseu- pathogenic bacteria are resistant to most antibiotics available in the domonas aeruginosa. Nevertheless, the number of pharmaceutical com- market and the mortality rate due to bacterial infections is over 2 - panies working on antibiotic research and the number of newly million per year, worldwide [1]. The older antibiotic classes discovered approved antibiotics dwindled sharply by 2010 [4]. The factors which more than 50 years ago — still in use after generations of synthetic led to only about 4 to 5 pharmaceutical giants to continue their antibac- tailoring — target a limited set of cellular pathways (e.g. cell wall/ terial research efforts are: antibiotic drug discovery is an expensive, slow protein/folate biosynthetic pathways) or macromolecular structures and uncertain process with an estimated cost of $800 M per drug devel- (DNA- and RNA-protein complexes, ribosomes and enzymes) [2]. oped, time span of 8 to 15 years and with a probability of approval of 1 in Given that for any new antibiotic that enters the market, resistance a 100 drug candidates (from discovery and preclinical phase to new is observed within years or even months, new drugs with a novel drug approval) [5–7]. Antibacterial drugs are not lucrative as they are mode of action are needed to avoid cross-resistance (i.e. resistance only used in patients for a short period of time till the infection is cleared. This contrasts with drugs for chronic diseases for which medication is continued through the rest of the patient's lifetime. Lastly, as frequently Abbreviations: SP(s), signal peptide(s); SPase I, Type I signal peptidase; Δ2–76 SPase I, quoted for antibiotics “the low hanging fruits are already picked” and so a truncated SPase I derivative devoid of the transmembrane segment residues; SPase II, Type II signal peptidase; SPase IV, Type IV signal peptidase; FRET, fluorescent resonance it is necessary to put in more effort and adopt novel strategies for finding energy transfer; MRSA, methicillin-resistant Staphylococcus aureus new antibiotics. Given the alarming increase in multidrug-resistant ☆ This article is part of a Special Issue entitled: Protein trafficking and secretion in pathogens, the dearth of new antibiotics and a dry drug-development bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. ⁎ pipeline, the problem is gaining wide attention and seeks urgent action Corresponding author. Tel.: +32 16337341; fax: +32 16337340. – E-mail addresses: [email protected] (S. Rao C.V.), [8 10]. A renewed interest in antibacterial research comes from bacteri- [email protected] (E. De Waelheyns), al genome analysis, uncovering ~300 highly conserved essential pro- [email protected] (A. Economou), [email protected] (J. Anné). teins that could serve as broad spectrum targets [11] in contrast to ~40

http://dx.doi.org/10.1016/j.bbamcr.2014.02.004 0167-4889/© 2014 Elsevier B.V. All rights reserved. S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1763 targets exploited so far by most commercial antibiotics [2]. However, 1.3.1. The Secretory (or the Sec-) pathway in bacteria finding new drugs against new targets brings in newer challenges [5,12]. The three primary stages in protein secretion are: targeting to the membrane, translocation across the membrane and the release of 1.2. What is required of an ideal new target? the mature protein into the external environment (in Gram-positive bacteria) or the periplasm (in Gram-negative bacteria) (Fig. 1a). ➢ Any novel antibiotic target should ideally be on the one hand essen- Gram-negative bacteria have additional branches (e.g. Type II and tial, conserved and universal in bacteria and on the other lacking or Type V) for protein export across the outer membrane. sufficiently dissimilar or non-essential in humans. Alternatively, tar- Proteins destined to function outside the cytoplasmic membrane gets that are not essential for growth but are required for virulence are generally synthesized as preproteins with a small N-terminal exten- or pathogenesis are also attractive as they do not impose a strong se- sion called the signal peptide (SP). Signal peptides, usually found at the lection pressure in bacteria that might lead to the development of re- amino terminus of secretory proteins, play a decisive role in targeting sistant strains. The idea behind anti-virulence targets is to attenuate and transport of preproteins. There are four kinds of signal peptides: the pathogen and facilitate elimination by the host immune system. Sec- and Tat-type SPs, known as Type I signal peptides, lipoprotein or ➢ The target gene product should be stable in vitro and amenable for Type II signal peptides, and Type IV or prepilin signal peptides [14]. high-throughput screens. Type I SPs have three recognizable regions namely a positively charged ➢ Specific targets with known function, structure and properties are amino-terminus (N-region), a central hydrophobic core (H-region) and desirable in initial drug-discovery efforts. a neutral, but polar C-terminus (C-region), as shown in Fig. 1b.The ➢ The cellular location of the target is an important factor as it should hydrophilic C-terminus contains the determinants for recognition be relatively accessible for a potential new drug. and processing by the signal peptidase. Type I SPs interact with ➢ It should be structurally and functionally distinct from a convention- components of the Sec-machinery (SecA, SecY and the signal al target or site or pathway to avoid cross-resistance. peptidases) [15] and except for Tat SPs (which are less hydrophobic), are thought to delay preprotein folding so as to retain translo- 1.3. Why target the bacterial secretion pathway? cation competence [16]. Type II SPs are similar to Type I SPs, but cleavage occurs immediately upstream of a Cys residue, and these More than 30% of bacterial proteins are destined to function at the are used to target lipoproteins. Type IV SPs differ from convention- cell envelope or outside the cell [13]. A vast majority of these proteins al N-terminal SPs in that they are usually very short and have no with functionally diverse roles such as nutrient uptake, excretion, me- classical tripartite structure [17,18]. tabolism, cell structure, communication, virulence and bacterial defense Targeting of secretory proteins via the Sec-pathway occurs post- are transported via the ubiquitous Secretion (Sec-) pathway. The Sec- translationally and in a poorly understood non-native state [19]. Some pathway is essential for viability, universally conserved in bacteria of the newly synthesized preproteins with export signals are recognized (as well as in the endoplasmic reticulum (ER) of eukaryotes and thyla- and escorted by secretion-specific chaperones, SecB, CsaA or by general koid membranes in plants) and is the primary route for protein export. chaperones, GroEL and/or DnaK [20].InE. coli Sec B helps in maintaining These features and the fact that some Sec-pathway components are not 20–30 preproteins in the unfolded, translocation-competent state and present in eukaryotes make it an attractive target. targets the bound preprotein to the membrane for translocation.

Fig. 1a. A schematic representation of the Sec-pathway in E. coli. Preproteins synthesized at the ribosomes are targeted to the membrane translocase, assisted by the cytosolic chaperone, SecB. The Sec-translocase consists of SecYEG protein conducting channel, accessory proteins SecDF (YajC, not shown) and the peripherally associated motor protein, SecA. SecA translo- cates the preprotein through the channel utilizing the energy from ATP hydrolysis. SPase I, a serine protease, cleaves non-lipoprotein substrates at the extracytoplasmic side, releasing the mature protein. SPase II is an aspartic acid protease which cleaves lipoprotein substrates beneath the extracytoplasmic membrane surface. SPase IV, also an aspartic acid protease, cleaves prepilins and pseudopilins at the cytoplasmic side of the membrane. YidC, a membrane protein insertase, together with the SecYEG channel, inserts membrane proteins via the SRP- pathway (not shown). The locations of the N- and C-termini of the membrane enzymes are indicated. The transmembrane helices are depicted as barrels. Figure adapted in part from [22]. 1764 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 b) SPase I cleavage site

-6 -3 -1 P/G P/G A x A N-region H-region C-region Mature protein

Fig. 1b. Tripartite structure of the Type I signal peptide. Bacterial Type I signal peptides typically contain a positively charged amino-terminal (N-) region, a central hydrophobic (H-) region and a neutral but polar carboxy terminal (C-) region containing the SPase I recognition sequence (AXA motif). Helix-breaking residues (Pro or Gly) are found in the middle of the H-region and at the boundary between the H- and the C-regions.

Translocation of the preprotein via the Sec-pathway (Fig. 1a)in- 2. The Type I signal peptidase (SPase I) as an antibiotic target volves (i) the SecYEG translocon which is comprised of three integral membrane proteins SecY, SecE and SecG, together forming the 2.1. SPase I: properties, structure, mode of action and its relevance as an an- protein-conducting channel. (ii) The peripherally associated motor pro- tibiotic target tein SecA, that drives the preprotein through the channel by repeated cycles of ATP binding and hydrolysis. (iii) The accessory heterotrimeric The physiological role of the Type I SPase (also known as “leader pep- complex SecDFYajC, which closely associates with SecYEG and is pre- tidase”) is to release the mature protein from the membrane for it to dicted to function at the late stage of translocation possibly by pulling reach its correct cellular or extracellular destination [28]. Without the preprotein from the periplasmic side or promoting its folding [21]. SPase I activity, the precursors remain membrane-bound due to the During or shortly after translocation, if applicable, the signal peptide uncleaved signal peptide acting as a membrane anchor [29]. Recent is removed by the signal peptidase (SPase), allowing the release of the studies indicate that the function of SPase I extends beyond secretion mature protein from the membrane. Among the preproteins, the major- and that they probably have a regulatory role as well. For example, the ity (non-lipoproteins) are processed by the Type I SPase (SPase I) while SPase I from S. aureus cleaves and inactivates lipoteichoic acid synthase lipoproteins are processed by the Type II SPase (SPase II). Precursors of (LtaS), a membrane embedded enzyme that synthesizes lipoteichoic pilin and related pseudopilins (Section 4.2) are processed by the Type IV acid, an important cell envelope component of Gram-positive bacteria SPase (SPase IV) [14,22]. Subsequently, the mature protein is folded at [30]. the trans side of the membrane. SPase I (EC 3.4.21.89) is an unconventional serine protease which does The Sec system (particularly the Sec YEG channel) along with a not use the canonical Ser/His/Asp triad but instead utilizes a Ser/Lys membrane protein insertase YidC, is also involved in membrane protein dyad and is classified into the evolutionary Clan SF, family S26 [31]. insertion but the targeting of these proteins occurs by the signal recognition particle (SRP) pathway (Section 4.3). In addition to the Sec pathway, several bacteria utilize the Twin arginine translocation (Tat) 2.1.1. Properties pathway for exporting a subset of proteins (Section 4.2), and although Gram-negative bacteria (e.g., E. coli, Salmonella Typhimurium) typi- there are differences in the targeting and the translocation apparati, cally have one chromosomally encoded SPase I which is essential and SPases I are common to both Sec and Tat pathways. constitutively expressed. One of the exceptions is P. aeruginosa, with two Type I SPases (LepB and PA1303) each with a distinct role in physiology and virulence [32]. Gram-positive bacteria (e.g., Bacillus subtilis 1.3.2. Antibiotic targets in the Sec-pathway and Streptomyces lividans) often have more than one SPase I, of which The integral membrane proteins SecY and SecE are essential for via- none of the individual enzymes is essential for cell viability on its own. bility and protein translocation while SecG is not essential but increases Exceptions include Streptococcus pneumoniae, Mycobacterium tuberculo- translocation efficiency [21]. The SecYEG proteins are universally con- sis and S. aureus [33]. SPases I are anchored to the membrane by one served, homologous to their eukaryotic counterparts Sec61 αγβ and (typically in Gram-positive) or more amino-terminal transmembrane have no enzymatic activity, thus are not ideal targets. Interestingly, indi- segments, (typically in Gram-negative bacteria). The carboxy-terminal rect targeting of SecY by some antibiotics [23,24] suggests that there is a catalytic domain is significantly smaller in SPases I of Gram-positive potential to exploit such an approach. SecD and SecF are not essential bacteria compared to those of Gram-negative bacteria. Despite these but improve the efficiency of translocation. The two proteins are not minor differences, SPases I contain five regions of high-sequence simi- present in eukaryotes but are not present in all prokaryotes either larity and identity referred to as boxes A to E, that are common and con- [25,26]. Although not broad-spectrum, SecD and SecF may be exploited served [29]. Box A consists of hydrophobic residues residing in the as targets as their deletion results in severely reduced protein secretion membrane spanning regions of the SPases I. Box B and Box D contain and growth and both proteins have large, soluble domains protruding the catalytic Ser and Lys residues respectively, while Boxes C and E con- on the trans side of the membrane, and would be accessible to potential tain residues important for substrate binding and for positioning of the drugs [26]. YajC (a protein of unknown function) is neither essential for catalytic residues. cell viability nor for protein translocation [21,27]. SPase I has substrate specificity for small and neutral residues at SPase I and SecA were the first Sec-pathway components pursued as − 3 (or P3) and − 1 (or P1) positions relative to the cleavage site targets. They are essential in bacteria and attractive as targets for which [34] (Fig. 1b). The SPase I recognition motif in the SP is referred to HTS assays exist and a few inhibitors are already available. Type II signal as ‘Ala-X-Ala’, as a consequence of the frequent presence of Ala residues peptidases, Type IV signal peptidases and YidC are potential candidates at these positions. During preprotein translocation, the N-region of that have also been pursued in recent years. the signal peptide acts as the topological determinant by following the In this review, we summarize the major developments, the current ‘positive-inside rule’ of membrane proteins [35]. The H-region is be- situation and progress of SPase I (Section 2) and SecA (Section 3)as lieved to span the membrane as an α-helix, while the C-region adopts antibiotic targets. We additionally briefly discuss the efforts towards a β-stranded conformation allowing access to the SPase I cleavage site targeting other components of the Sec-pathway as well as the other on the periplasmic side and processing [36]. The signal peptides pathways associated with bacterial protein secretion (Section 4). in Gram-negative bacteria are generally shorter than those in Gram- S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1765 positive bacteria. In case of the former, where the signal peptide is not and an extended β-ribbon are mostly limited to Gram-negative bacteria long enough to span the membrane, the H-region is believed to change [36]. An unusually large hydrophobic surface that includes the catalytic from an α-helix to an extended structure in the membrane [22].Apart center and the adjacent substrate binding sites is the proposed mem- from Sec substrates, SPase I processes preproteins with Tat signal pep- brane association surface (Fig. 2). This surface, predicted to be in close tides (Section 4.3) and a few membrane proteins [22]. An interesting association with the membrane and capable of inserting into the lipid example is the membrane-embedded enzyme LtaS (Section 2.1) with bilayer during signal peptide cleavage, explains the detergent require- five N-terminal transmembrane helices in which the Ala-X-Ala motif is ment of the SPase I for optimum activity and crystallization [36]. Two located not after an N-terminal signal peptide but 44 residues after the shallow hydrophobic depressions appropriate for accommodating last transmembrane domain [30]. For more details about the signal pep- small aliphatic residues form the substrate binding pockets S1 and S3 tide processing by SPases I, see Paetzel M [37]. (Fig. 2) and provide the basis for Ala-X-Ala specificity. Site-directed mu- The biochemical properties of E. coli LepB as well as SPases I from tagenesis identified two Ile residues (Ile87 and Ile145, E. coli number- Gram-positive bacteria B. subtilis, S. lividans, S. pneumoniae,(seereview ing) in the borderline between the S1 and S3 sites, contributing to the [33]), and S. aureus [38] have been studied in detail. The common high fidelity and substrate specificity of the SPase I [46]. in vitro properties of SPase I include: (i) concentration and time- Paetzel et al. [14,34,37] proposed the proteolytic mechanism of the dependent, intermolecular-self cleavage leading to inactive fragments, bacterial SPase I which is illustrated based on current data. Briefly, the (ii) optimum activity requires non-ionic detergents (such as Triton-X- SP binds to the SPase I making β-sheet type hydrogen bonding interac- 100), (iii) phospholipids enhance activity and (iv) optimum pH varies tions with both β-strands that line the substrate binding sites of the from pH 8 to pH 11, in contrast to classical serine proteases. SPase I [44]. The P1 and P3 residues of the SP occupy the S1 and S3 bind- E. coli LepB is the best characterized among the SPases I [34]. A trun- ing pockets of the SPase I, respectively, while P5 and P7 residues occupy cated derivative, Δ2–76 SPase I (previously Δ2–75) lacking residues potential binding sites S5 and S7, respectively [45]. P2 is solvent ex- 2–76 corresponding to the transmembrane segment was designed, pu- posed which explains the lack of specificity at this position. Upon sub- rified and characterized in vitro [39,40] (Note that the E. coli numbering strate binding, the -amino group of Lys146 (general base) abstracts a was changed recently [41] owing to a missing residue in the sequencing proton from the side chain hydroxyl group of Ser91 (nucleophile), acti- data. The results remain unaffected as the missing residue is from the vating the Ser91 Oγ for si-face attack on the carbonyl of the scissile bond cytoplasmic loop region, only the numbering used in the previously re- (SPase I cleavage site). The resulting tetrahedral intermediate I is stabi- ported crystal structures are now up by one number). The Δ2–76 SPase I lized by an oxyanion hole formed by the Ser91 main chain amide hydro- is active in vitro, but less efficient compared to the full-length SPase I gen and the Ser89 side-chain hydroxyl hydrogen group. The ammonium [42]. Crystal structures of the Δ2–76 SPase I in its Apo-form [40,43] group from the side chain of Lys donates a proton to the new amino- and in the complex with inhibitors [36,41,44,45] have helped in under- terminus of the now mature protein and the tetrahedral intermediate standing the unique properties of the enzyme. breaks down to produce signal peptide bound acyl-enzyme. Lys146 acts as a general base again by activating the deacylating water, which 2.1.2. Structure and mode of action forms the tetrahedral intermediate II. The Lys146 side-chain ammoni- Crystal structure of E. coli LepB Δ2–76 (Fig. 2) has a unique mostly um group donates a proton to the Ser91 Oγ, resulting in the dissociation β-sheet protein fold, consisting of two antiparallel β-sheet domains, of the signal peptide and regeneration of the SPase I active site. domain I and II. Domain I contains all the conserved regions (boxes B– E) in bacterial SPase I, while domain II (an insertion within domain I) 2.1.3. Relevance as antibiotic target SPases I are attractive targets because: (i) they are ubiquitous and conserved in bacteria. (ii) SPase I activity is essential for growth and viability in bacteria. This was first demonstrated by genetic [47] and functional studies [48] in E. coli. SPase I inhibition leads to accumulation of preproteins in the cell membrane resulting in cell death [49]. (iii) SPases I differ sufficiently from their eukaryotic counterparts (present in ER and mitochondria of humans) in structure, locali- zation and catalytic mechanism, thereby reducing possibilities for toxicity [14]. Unlike bacterial SPases I which are monomeric, eukaryotic SPases are multimeric complexes, located in the inner membrane of mitochondria or in the lumen of the ER and in the case of the latter, operate by the conventional Ser/His/Asp triad mechanism or Ser/His dyad mechanism. (iv) The Ser/Lys catalytic dyad mechanism and the si-face attack of its substrates (rather than re-face attack used by most Ser- dependent proteases), characteristic of the SPase I allows selec- tive target inhibition without inhibiting other essential proteases in eukaryotes. (v) The catalytic domain of the bacterial SPase I, exposed to the outer leaflet of the plasma membrane, is accessible to potential inhibitors. Fig. 2. Structure showing SPase I bound to inhibitors. Structure of the catalytic domain of (vi) SPase I inhibition is likely to result in simultaneous attenua- β E. coli SPase I in complex with (i) 5S-penem inhibitor and (ii) arylomycin A2/ -sultam in- tion due to its role in virulence. hibitors (superimposed). SPase I is shown as white molecular surface with the catalytic dyad Ser/Lys highlighted in red. The 5S-penem inhibitor is shown in blue ball-and-stick, Molecular and functional analyses of the gene encoding SPase I β the arylomycin A2 inhibitor is shown in magenta ball-and-stick, and the -sultam inhibitor in several bacteria including human pathogens such as S. Typhimurium is shown as yellow ball-and-stick. A bound fragment of detergent, Triton X-100 is shown [50], S. aureus [51], S. pneumoniae [52], Rickettsia rickettsii [53], in orange ball-and-stick. The approximate positions of the proposed substrate binding sites and the membrane association surface are indicated. Legionella pneumophila [54], S. epidermidis [55], P. aeruginosa [32] and Figure reproduced with permission from [45]. M. tuberculosis [56] have confirmed its essential role. 1766 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

SPase I is a suitable target against resistant pathogens requiring ur- Table 1 gent medical intervention: SPase I (in vitro) activity with the two best substrates. Substrates Speciﬁc enzymatic activity Reference S. aureus, responsible for a wide array of infections (including skin, (M−1 s−1) bone, lung and blood stream) and known for its remarkable ability Escherichia Staphylococcus to develop resistance, has one active and essential SPase I, termed coli LepB aureus SpsB

SpsB. The enzyme is characterized in vitro and validated as a target Preprotein substrate [38,51]. Several SpsB-dependent proteins required for virulence Pro-OmpA-nucleaseAa 1.1 × 107 16 [65] such as extracellular proteases, lipases, superantigens, haemolysins Synthetic substrate ↓ 5 6 and leukocidins were identified by secretome profiling of S. aureus Decanoyl-LTPTAKA ASKIDD-OH 4.2 × 10 2.3 × 10 [67] [57]. SpsB also plays a role in the agr quorum sensing system, Amino acids are abbreviated as single letter codes. The arrow indicates the point of which is important for biofilm formation and virulence [58]. SpsB cleavage and P1 and P3 residues are in bold. a is also suggested to have a regulatory role due to its proteolytic ac- A hybrid protein made up of signal peptide of outer membrane protein A from E. coli and the nuclease A from S. aureus. tion resulting in inactivation of LtaS, as discussed in Section 2.1. P. aeruginosa, an opportunistic pathogen, naturally resistant to many frontline antibiotics, has two SPases I, LepB and PA1303, character- 2.2.1.2. Fluorescent resonance energy transfer (FRET)-based assays. Syn- ized recently. LepB is the essential and primary SPase I, while thetic substrates for SPase I are short stretches of residues, identical PA1303 is non-essential with a likely function in the suppression of to those in the signal peptide region of preproteins containing the virulence factor secretion through the quorum-sensing (QS) cascade SPase I recognition and cleavage site. Peptides that are at least 9-mers [32]. In silico analysis predicts 801 P. aeruginosa proteins (14.4% of and in addition have SP-like features incorporated in them are far better the genome) as containing putative SPase I substrates. The sub- substrates. For example, Stein et al. [66] appended K5-L10 to incorporate strates include components of the Sec-pathway, outer membrane a positively charged N-terminus and a hydrophobic core, respectively proteins and porins, flagellar structural proteins, cell wall biosynthe- (see Table 2, substrate 1) whereas Bruton et al. [67] incorporated a sis enzymes as well as virulence factors including elastases (LasA decanoyl moiety (mimicking a membrane anchor) and a turn inducing and LasB), endotoxin A, β-lactamase and proteins involved in bio- motif (proline at P5 position) as shown in Table 1 (substrate 2) and synthesis of alginate, suggesting a direct role in house-keeping as Table 2 (substrate 4). well as pathogenesis [32]. A difference in substrate specificity of SPases I is observed even with S. pneumoniae, responsible for considerable illness (including pneu- the synthetic substrates (see review [68]). Thus in vitro inhibition monia, ear infections and meningitis) and mortality due to drug- assays developed for SPases I from Gram-negative E. coli and Gram- resistant strains, has one SPase I, Spi. It is also a well characterized positive pathogens, S. pneumoniae, S. aureus and S. epidermidis, involve and validated target [52,59–61]. specifically designed substrates (see Table 2). The in vitro assays facili- Listeria monocytogenes, a food-borne pathogen, has three paralogs of tated the characterization and comparison of SPases I from different SPase I, SipX, SipY and SipZ. Deletion of the sipZ gene (encoding the bacteria. Often these assays are FRET-based, allowing rapid measure- major SPase I) results in impaired secretion of virulence factors (e.g., ment and some are already optimized for high-throughput screening listeriolysin O and phospholipase C), rendering it almost avirulent of inhibitors. The in vitro inhibition assay for S. pneumoniae was used [62], highlighting the function of SPase I in virulence. for high-throughput screening of 50,000 pre-fractionated natural S. epidermidis, a common cause of nosocomial infections and product samples [59], of which only one sample inhibited SPase I. This fi bio lm-associated foreign body infections has two active SPases I was identified as lipoglycopeptide or Arylomycin C (Section 2.3.3). We (Sip2 or SpsIB and Sip3) which are attractive targets not only in optimized a high-throughput FRET-based assay for S. aureus SpsB and fi free-living but also in bio lm-associated S. epidermidis cells [55]. screened ~26,000 diverse small molecules (average Z′ value [69] of 0. Consistent with its role in pathogenesis, secretome analysis of 73) but did not obtain potent hits [38,70].Z′ scores are measures of SPase I-dependent proteins during stationary growth phase of standard deviation, derived using the formula: S. epidermidis, identified 11 proteins including peptidoglycan hydrolases, proteases and lipases, which are important for virulence [63]. ÀÁ 3σ þ þ 3σ − M. tuberculosis has one essential Type I SPase, LepB [56]. LepB is a Z Prime or Z′ ¼ 1− c c μ −μ promising target as demonstrated using an SPase I inhibitor (MD3, cþ c− a beta-aminoketone; see Supplementary Fig. S1 online) which is bac-

tericidal against actively growing cells and even more potent against where, σC+ and σC− are standard deviations of positive and negative non-replicating or persistent cells [56]. However, the MD3 inhibitor controls, respectively while μC+ and μC− are the means of positive and is not suitable for drug development due to compound instability. negative controls, respectively. The in vitro efﬁcacy of a rationally designed peptide aldehyde inhib- 2.2. SPase I inhibition assays itor (Section 2.3.2) was demonstrated using the SpsB FRET assay [71].

The entire range of SPase I assays used at different stages of inhibitor 2.2.2. In vivo assays search is summarized below. The secretion of β-lactamase, a Sec-dependent substrate, is used to gauge in vivo SPase I activity/inhibition. Initially, whole cell pulse- 2.2.1. In vitro inhibition assays chaseanalysisofprocessingofpre-β-lactamase to β-lactamase in E. coli was used to demonstrate the inhibition of preprotein processing 2.2.1.1. Preprotein processing assay. This assay involves incubation of the in situ by a SPase I inhibitor, penem [72]. This test is now replaced by purified SPase I and preprotein followed by separation of products by the β-lactamase secretion assay. SDS-PAGE [64]. Pro-OmpA-Nuclease A, a hybrid protein of S. aureus nuclease A fused to the signal peptide of E. coli outer membrane protein A 2.2.2.1. β-lactamase secretion assay in S. aureus. The assay involves mea- (OmpA) [34,65], is an excellent substrate for measuring in vitro activity surement of hydrolysis of nitrocefin, a chromogenic β-lactamase sub- as well as for kinetic analysis of SPase I (Table 1). The assay is gel-based strate in culture supernatants incubated in the presence and absence and as such not ideal for HTS of inhibitors although it can be used as a of an inhibitor. The assay is used as a secondary assay for SPase I inhibi- confirmatory assay for SPase I inhibition [38]. tion [59,73]. S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1767

Table 2 SPase I substrates for rapid screening of inhibitors in FRET assays.

Sl. no. Synthetic peptide substrate Preprotein used for peptide design Microorganism/SPase I (kcat/KM)Reference sequence a

1. K(5)-L(10)-Y(NO2)FSASALA↓KIK(Abz) Maltose binding protein from Escherichia coli E. coli LepB [66] (2.5 × 106 M−1 s−1) 2. KLTFGTVK(Abz)PVQA↓IAGY(NO2)EWL Streptokinase from Streptococcus pyogenes S. pneumoniae Spi [61] (2.7 × 102 M−1 s−1) 3. (Dabcyl)-ADHDAHA↓SET-(EDANS) SceD from Staphylococcus aureus S. aureus SpsB [38] (1.85 × 103 M−1 s−1) ↓ 4. Decanoyl-k(Dabcyl)-TPTAKA ASKKD-D(EDANS)-NH2 Consensus preprotein cleavage sites in S. aureus SpsB [73] S. aureus 5. (Dabcyl)VSPAAFA↓ADL(EDANS) LasB elastase from Pseudomonas aeruginosa P. aeruginosa LepB and PA1303 [32]

a Amino acids are abbreviated as single letter codes. The arrow indicates the point of cleavage and P1 and P3 residues are in bold.

2.2.3. Target-based whole cell assays β-lactam inhibitors of SPase I: monocyclic azetidinones, the first (non- These have an advantage over the assays mentioned above as com- peptide) inhibitors effective against E. coli LepB albeit at very high con- pounds are tested based on both the mode of action as well as the anti- centrations (500 μM) [78];and5 S-Penem stereoisomers, the first po- bacterial activity. These assays are based on the observation that tent irreversible inhibitors [79] (Table 3, inhibitor 1). Extensive reduced expression of an essential target gene renders the strain sensi- in vitro screening and optimization efforts at GlaxoSmithKline tive to compounds that inhibit that particular gene product or pathway. (previously SmithKline Beecham) [72,77,80,81] resulted in a few potent inhibitors including allyl (5S, 6S)-6-[(R)-acetoxyethyl]-penem-3- μ 2.2.3.1. Regulated expression of SPase I. A cellular assay employing an carboxylate (Fig. 3), with an IC50 of 0.38 M against E. coli LepB. Based E. coli strain underexpressing lepB gene was validated for testing SPase on the 5S-stereochemistry of the inhibitor, it was proposed that SPase I inhibitors using penem, a known SPase I inhibitor [74]. The strain has I attacks the peptide backbone of its substrates from the si-face (uncom- E. coli lepB under an arabinose regulatable promoter cloned onto a plas- mon for serine-dependent hydrolases) rather than from the re-face. mid while the chromosomal lepB gene has been removed. The assay was The inhibitor was co-crystallized with the catalytic domain of the used as a secondary assay to test three potential inhibitors derived from SPase I to obtain a high resolution structure that provided a direct HTS in vitro. The inhibitors did not have significant antibacterial activity proof of SPase I utilizing the Ser/Lys catalytic dyad mechanism [36]. against E. coli [74]. Recently, one of the inhibitors, MD3, was reported to The structure showed that Ser 91 (E. coli numbering) attacks the si- face of the β-lactam amide bond which is a peptide bond analog. The be active against M. tuberculosis (MIC99 of 17.7 μM) [56] (Section 2.1.3). In M. tuberculosis,alepB overexpression strain showed reduced sus- crystal structure by Paetzel et al. [34,36] provides valuable information ceptibility to the aforementioned β-aminoketone inhibitor while the on the active site, helps explain the unique characteristics of the enzyme lepB underexpression strain showed increased susceptibility to the in- and serves as a template for inhibitor design. Despite high in vitro activ- hibitor, validating SPase I as the target [56]. ity, the penem-type inhibitors (Fig. 3), generally have poor antibacterial activity (Table 3, inhibitors 1 and 2) likely due to low cell wall penetration or compound instability [77,82], limiting their clinical 2.2.3.2. Bacterial target array -S. aureus fitness test (SaFT) profiling. This is application. A couple of exceptions noted in a recent study are that a method to determine the mode of action of a new compound by si- penem and a carbamate-derivatized penem are moderately effective multaneous screening against all essential bacterial gene targets. It is against S. epidermidis and MRSA, respectively [83] (Table 3, inhibitors based on 245 S. aureus antisense RNA strains, each of which is 3and4). engineered for reduced expression of (conserved, broad-spectrum) target genes essential for its growth [75].Briefly, this semi-automated 2.3.2. Designing peptide, lipopeptide and peptide aldehyde inhibitors method involves growth of strains in the presence and absence of test Early studies proved that (i) SPases I are competitively inhibited by compounds, determination of relative abundance of the strains by mul- signal peptides in vitro [84]. (ii) Preproteins or synthetic substrates tiplex PCR, capillary electrophoresis and DNA fragment analysis to with Pro at +1 position inhibit SPase I activity in vivo, leading to an im- quantify strain-specificmarkers[75,76]. This strategy adopted by paired growth of cells [85,86]. Attempts at designing peptide inhibitors Merck resulted in finding several new classes of natural compounds based on the classical approach used for serine protease inhibitors did [11] including inhibitors for the Type I signal peptidases [73]. not work initially for the SPase I [77] till researchers at GlaxoSmithKline finally came up with highly effective lipopeptide inhibitors [67] and a 2.3. Overview of SPase I inhibitors substrate for in vitro inhibition assay (Table 1, substrate 2). The peptides were based on the consensus sequence of preproteins in S. aureus and SPases I are insensitive to classical protease inhibitors including ser- designed to incorporate key structural elements of a signal peptide, ine protease inhibitors [34]. Based on the observation that there is at namely an alkyl membrane anchor, a helix breaking Pro at P5 and a least one other enzyme (signal peptide peptidase A) operating with Lys at P2 (for the AKA). Insertion of Pro at +1 converted the peptide the same Ser-Lys catalytic dyad mechanism that is inhibited by common into a competitive inhibitor while incorporation of α-ketoamide at the serine protease inhibitors, Dalbey et al. [22] suggested that serine prote- cleavage site transformed it to a time-dependent inhibitor of S. aureus ase inhibitors do not bind with high affinity to SPase I. SpsB (IC50 of 0.6 and 0.1 μM, respectively) [67] (Table 3, inhibitors 5 The search for effective inhibitors for SPase I, a challenging task from and 6). The antimicrobial activity of the lipopeptide is undisclosed. Fur- the start [77] has led to some exciting developments as illustrated ther optimization resulted in a lipopeptide aldehyde inhibitor (Fig. 3) below. with increased potency against SpsB (IC50 of 0.09 μM) but with poor antimicrobial activity [71] (Table 3, inhibitor 7). These studies proved the 2.3.1. Discovery of Penems (β-lactam type inhibitors) critical role of N-terminal fatty acid for potency of the inhibitor in vitro Although the essential role of SPase I in E. coli was demonstrated in as well as in vivo. It is presumed that it probably mimics the hydropho- the early eighties [47,48], the mechanism of action of SPases I remained bic region of the signal peptides in preproteins. This also explains why unclear till the late nineties. Two pharmaceutical companies discovered peptides as such did not prove to be effective inhibitors. Interestingly, 1768 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Table 3 SPase I inhibitors with their in vitro and in vivo activities.

a Sl. no. Inhibitor SPase IC50 (μM) Microorganism MIC (µM) Reference Penems 1. Allyl (5S,6S)-6-[(R)-acetoxyethyl] penem-3-carboxylate LepB 0.38 Escherichia coli – [77] 2. (5S)-Tricyclic penem LepB 0.2 E. coli – [82] SpsB 5 MRSA N272 3. (5S,6S)-6-[(R)-hydroxyethyl] penem core with a E. colib ≥596 [83] C3 p-nitrobenzyl protected carboxylic acid Staphylococcus aureus ≥596 Staphylococcus epidermidis 149 4. Carbamate-derivatized penems (ethyl) MRSA 247 [83] (isopropyl) 238

Rationally designed peptides/lipopeptides 5. Decanoyl-LTPTAKAPSKIDD-OH SpsB 0.6 S. aureus – [67] 6. Decanoyl-LTPTANA-α-ketoamide analogs SpsB 0.1–16 S. aureus – [67] 7. Decanoyl-PTANA-aldehyde SpsB 0.09 S. aureus 205 [71] LepB 13.4 E. coli N819

8. (D)-KLKL6KLK-NH2 LepB 30 E. coli 16 [87] E. colib 4.8 S. aureus 8 Streptococcus pneumoniae 4.8 Haemophilus inﬂuenzae N64

Arylomycins c 9. Arylomycin A2 LepB – E. coli (P) N128 [91–93] S. aureus(P)c N128 Pseudomonas aeruginosa (P)c N128 S. epidermidis (S)c 1 d c 10. Arylomycin A-C16 (synthetic arylomycin A2 S. hominis (S) 0.25 derivative with a longer fatty acid tail) Corynebacterium glutamicum (M)c 2 Rhodococcus opacus (V)c 2 Helicobacter pylori (A)c 4 S. pneumoniae (N)c 16 S. pyogenes (A)c 16 11. Arylomycin C (Lipoglycopeptides) LepB 0.11–0.19 E. coli 4–8 [59] 2.4–24.9 S. pneumoniae 8–64 Spi S. aureus 32–64 H. inﬂuenzae ≥64 12. Actinocarbasin (Arylomyicn D) SpsB 0.05 MRSA 5.9 0.3+ IPMe [73] MRSA strain COL 13. M131 (synthetic derivative of Actinocarbasin) SpsB 0.01 MRSA 1.2 0.15 + IPMe [73] MRSA strain COL

Krisynomycin 14. Krisynomycin SpsB 0.12 MRSA 46.2 2.9 + IPMe [73] MRSA strain COL

MIC: Minimum inhibitory concentration. a Half-maximal (50%) inhibitory concentration. b Polymyxin B nonapeptide permeabilized cells. c Resistance-conferring Pro or other residues at analogous position (see text), denoted as amino acid single letter code. d Arylomycin A-C16 was formerly called arylomycin C16. e Imipenem (IPM) susceptibility (MIC = 13.4 μM) restored by SPase I inhibitors. a signal peptide mimic designed and synthesized based on an anti- Arylomycins are secondary metabolites from Streptomyces spp., microbial peptide from Sacrophaga peregrina (ﬂesh fruit ﬂy) inhibits synthesized by nonribosomal peptide synthesis. They are lipohexa-

E. coli LepB (IC50 of 30 μM) and has antibacterial activity against E. coli peptides with a common core structure consisting of a [3,3] biaryl- (MIC = 16 μM), S. aureus (8 μM) and S. pneumoniae (4.8 μM) [87] linked tripeptide macrocycle at the C-terminus and a fatty acid tail (Table 3, inhibitor 8). attached to the N-terminus (Fig. 3). Arylomycin B series differ from A by nitro-substitution of the tyrosine residue (Fig. 3). Arylomycin C series 2.3.3. Discovery of natural product Arylomycins differ from the A series by glycosylation, and in some cases hydroxyl- Arylomycins are among the most extensively studied SPase I inhibi- ation of the macrocycle and a longer fatty acid tail [59,90] (Fig. 3). tors as they are the first inhibitors with significant antibacterial activity. Arylomycin A2 does not inhibit the growth of E. coli, P. aeruginosa and There are currently four related families of arylomycins (including S. aureus, while it is highly potent against S. epidermidis (MIC of 1 μg/ml) actinocarbasin, Section 2.3.4) identified by independent groups. [91] (Table 3, inhibitor 9). In an interesting study, Romesberg et al. [92] Arylomycins (A and B series) were first reported in 2002 with demonstrated that S. epidermidis develops resistance to arylomycin by a their structure and limited antibacterial activity [88,89]. Subsequently, point mutation (Ser to Pro at residue 29) in SpsIB, one of the two active the crystal structure of Δ2–76 SPase I in complex with a member, SPases I. The resistance-conferring Pro is responsible for natural resis-

Arylomycin A2, revealed their mechanism of action [44]. Lipogly- tance to arylomycins in bacteria such as E. coli, P. aeruginosa and in copeptides (Arylomycin C series) were identiﬁed in parallel (by Eli some strains of S. aureus. The absence of Pro is often predictive of Lilly and Company) as inhibitors of E. coli and S. pneumoniae SPases I arylomycin sensitivity with only a few exceptions [92] (Table 3,inhibi- in a high-throughput FRET-based assay (Section 2.2.1.2) [59]. Modest tor 10). The otherwise broad-spectrum activity of arylomycins was antibacterial activity was observed against a few pathogens [59] demonstrated using a synthetic derivative with a longer fatty acid tail

(Table 3, inhibitor 11). (Arylomycin A-C16) [92]. In fact its potency against a few coagulase- S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1769 negative staphylococci (Staphylococcus haemolyticus, Staphylococcus are natural antibiotics for which resistance has evolved and so their hominis, Staphylococcus lugdunensis) is equal to or greater than that of broad-spectrum activity is more likely to be restored by synthetic tailor- vancomycin, commonly prescribed for treatment of MRSA [93]. ing compared to molecules that have never been antibiotics [90]. The The molecular basis for resistance was provided by the crystal struc- crystal structure data provides additional possibilities for optimizing ture of arylomycin in complex with the E. coli SPase I [44] which shows the arylomycin scaffold. that the resistance-conferring Pro residue is present in a less conserved Arylomycin A2 binds non-covalently to the SPase I in a manner region of the SPase I (away from the catalytic center but within thought to mimic its natural substrates. The inhibitor position in the the substrate binding pocket) with which arylomycin interacts [92]. binding pocket with its C-terminal tripeptide macrocycle points The mutation reduces the binding afﬁnity of arylomycin with the towards the active site and makes a parallel β-strand interaction with β- SPase I. Chemical synthesis of arylomycins (A, B and C series) and strand that lines the SPase I binding cleft. The methylene groups of the their derivatives have facilitated structure–activity relationship (SAR) C12 fatty acid make Van der Waals interaction with the predicted mem- studies (see review [90]). Synthetic arylomycin derivatives (with meth- braneassociationsurfaceoftheSPaseI.Thisprovidesarationaleforthe ylene units in the arylomycin backbone) designed to compensate for the critical role of amino-terminal fatty acid for effectiveness of SPase I unfavorable interactions caused by the resistance-conferring Pro, re- inhibitors and substrates. SAR studies in arylomycin indicate that a mini- stored some activity against wild-type S. aureus [90]. Thus, arylomycins mum of C12 tail is required and those up to C16 are ideal [90].Thecrystal

Fig. 3. Chemical structures of SPase I inhibitors indicated in Table 3. 1770 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Fig. 3 (continued).

structure of arylomycin in ternary complex with the SPase I and a weak in- A study used model strains of E. coli and S. aureus (with a point mu- hibitor, morpholino-β-sultam derivative (BAL0019193; see Supplementa- tation to remove the resistance-conferring Pro) to demonstrate that the ry Fig. S1 online) also suggests a scope for further optimization. The β- antibiotic activity of arylomycins is a result of insufficient SPase I activity sultam derivative binds noncovalently at the active site, taking a parallel and it can be either bacteriostatic or bactericidal, depending on the orientation relative to the biaryl ring moiety of arylomycin, defining a organism and growth conditions [95]. In addition, arylomycins show novel space that can be occupied by optimizing the arylomycin scaffold relatively little synergy or antagonism with most other antibiotic classes [45] (Fig. 2). The structure of SPase I in complex with a lipoglycopeptide with the exception of gentamicin, an aminoglycoside, with which it (arylomycin C) derivative shows that the sugar moiety is directed away shows synergy (Table 4). from the binding site, suggesting that other modifications may be made at this position to possibly optimize the pharmacokinetics of the scaffold 2.3.4. Discovery of actinocarbasin and krisynomycin [41]. A recent study showed that C-terminal homologation with a glycyl Actinocarbasin and krisynomycin are structurally distinct, natural aldehyde and the addition of a positive charge to the macrocycle increase products with significant antimicrobial activity, identified by a reverse the activity and spectrum of arylomycin [94]. genomic approach [73]. This approach (also sometimes referred to as S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1771

Fig. 3 (continued).

compound-driven target identification, chemical biology, or chemical (Table 4). Thieren et al. [73] demonstrated that the synergy of the genetics), is to screen for compounds with antibacterial whole cell ac- SPase I inhibitors is specifictotheβ-lactam class (including penicillins, tivity and then determine their mechanism of action using various bio- cephalosporins and carbapenems, except for ceftazimide) and not for chemical and genetic approaches. Imipenem (a carbapenem) is a highly any other classes of antibiotics tested and specific for MRSA. Synergy successful β-lactam antibiotic which is ineffective against MRSA. To of M131 with imipenem is also effective in vivo, in murine models of find potentiators of imipenem (a second agent added to fight specific MRSA infection; and importantly, combining imipenem with SPase I in- resistance to imipenem) against MRSA, a high-throughput, MRSA agar hibitor M131 suppresses the emergence of M131 resistance. growth assay was used to screen molecules derived from microbial ex- β-lactams are the most successful antibiotics for treatment of bacte- tracts [73]. This led to identifying krisynomycin (a cyclic depsipeptide) rial infections caused by numerous species, spanning 60 years and isolated from Streptomyces fradiae strain MA7310 and actinocarbasin representing over 65% of the world antibiotic market [96]. Given that, (a lipoglycopeptide) from Actinoplanes ferrugineus strain MA7383. The combination therapy is expected to account for the most common use two molecules potentiate the activity of imipenem at a concentration of the β-lactams in future [96], the synergy of β-lactam antibiotics with 16-fold below their native MIC value (Table 3, inhibitors 12 and 14). SPase I inhibitors holds the potential to broaden the spectrum of An optimized synthetic derivative of actinocarbasin, M131 (Fig. 3) β-lactams against MRSA and also reduce the emergence of resistant strains. has improved potency and synergy with imipenem (Table 3, inhibitor 13). The cellular target of actinocarbasin, M131 and krisynomycin 2.3.5. Potential SPase I inhibitors from in silico screens was identified as the SPase I, SpsB, by S. aureus fitness test profiling In M. tuberculosis, although targeting LepB might potentially lead to (Section 2.2.3.2). Their potency was confirmed in a FRET-based assay eliminating persistent bacteria and shorten therapy, neither compound (Section 2.2.1.2)andβ-lactamase secretion assay (Section 2.2.2.1). MD3 (used for target validation [56]) nor arylomycin (due to the pres- Actinocarbasin has the same core macrocycle as the arylomycins but ence of resistance conferring Pro in its SPase I) can be currently pursued. with O-sulfonation on the aryl rings and extensive modification of the Recently, a high-throughput virtual screening of 169,109 natural lipopeptide tail (Fig. 3), and hence recently grouped into the arylomycin compounds (from the ZINC database; http://zinc.docking.org) using a Dseries[90]. Krisynomycin (Fig. 3) belongs to a new structural class. model of the 3D structure of the M. tuberculosis SPase I, resulted in the Synergy of the molecules with imipenem against MRSA clinical identification of two different inhibitors EMP and PHM (see Supplemen- isolates was demonstrated using the checkerboard method [73] tary Fig. S1 online), which bind to the active site of the enzyme in silico 1772 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Table 4 Synergy of SPase I inhibitors with Imipenem (β-lactam) and Gentamicin (an aminoglycoside) determined by checkerboard analysis.

Antibiotic + SPase I inhibitor FIC index rangea Microorganism Reference

Imipenem + Actinocarbasin 0.19–0.42 Clinical isolates of MRSA [73] Imipenem + Krisynomycin 0.15–0.26 Clinical isolates of MRSA [73] b c d Gentamicin + Arylomycin A-C16 0.55 ± 0.11–0.91 ± 0.25 Escherichia coli PAS0260 [95] b c d Gentamicin + Arylomycin A-C16 0.46 ± 0.12–0.83 ± 0.22 Staphylococcus aureus PAS8001 [95] a FIC: Fractional inhibitory concentration; FIC of a test agent in a particular combination is the MIC of that agent in the combination divided by the MIC of that agent when tested alone. FIC index is the sum of the FICs of the two antibiotics. FIC indexes of ≤0.5 reflect significant synergism, while FIC indexes ≥2reflect significant antagonism. b A synthetic derivative of arylomycin A2, with a longer fatty acid tail. c Averages of minimum and maximum FIC values observed, respectively ± standard deviations. d Mutant strains which differ from the WT, by a point mutation that converts resistance conferring Pro to Leu or Ser, for increased arylomycin sensitivity.

[97]. Both EMP and PHM form hydrogen bond interactions with residues is involved in signal peptide binding [116,118,119] and the expandable in the active site and substrate binding pockets. PHM forms a hydrogen groove 2 (known as “the clamp”), created at the interface of PBD and bond with the nucleophilic Ser. The compounds are yet to be validated IRA2, was proposed to bind mature regions of the preprotein [104]. in vitro and for their antibacterial activity against M. tuberculosis. Large-scale conformational changes of the PBD are expected to be required for initiation of the translocation process [120]. SecA is a flexible enzyme, a property that aids its interaction with 3. SecA as an antibiotic target multiple ligands and also its motor function [104,112,121]. SecA interacts with nucleotides, preproteins, signal peptides [118],SecB[122] 3.1. SecA: properties, structure and mode of action; relevance as an antibi- and other chaperones, ribosomes [123] as well as other components otic target and druggable sites (see below), all of which influence its activity [112]. Briefly, anionic phospholipids (phosphatidylglycerol and cardiolipin) are important 3.1.1. Properties, structure and mode of action for SecA binding to the membrane while nonlamellar-prone lipids SecA is a mechanoenzyme with an N-terminal ATPase domain and (diacylglycerol and phosphatidylethanolamine) specifically enhance a C-terminal specificity domain (MW 102 kDa, in E. coli). The crystal SecA binding to the bilayer. SecA binds to the membrane with low affin- structures of SecA from E. coli [98,99], M. tuberculosis [100], B. subtilis ity at acidic phospholipids and with high affinity (low nM) at the [101–105], T. maritima [103,104], T. thermophilus [106], show a common SecYEG channel [112]. Binding of SecA to the various ligands and finally structural and functional domain organization (Fig. 4). to the SecYEG-SecDFYajC complex allosterically stimulates the ATPase SecA is classified into the superfamily II (SF2) DEAD (DExH/D) pro- activity of SecA for driving the translocation process. teins, which includes nucleic acid helicases [107,108]. Functionally, SecA-mediated targeting and secretory protein translocation across helicases utilize energy from ATP hydrolysis to bind and unwind nucleic the E. coli inner membrane is detailed in Chatzi et al., in press (this vol- acid substrates, while SecA uses this chemical energy to bind and mobi- ume). The mode of action of SecA, based on existing biophysical and lize polypeptide substrates through the protein conducting channel. biochemical data [108,113], is briefly outlined here: cytosolic SecA Structurally, both SecA and helicases contain two RecA-like subdomains with Walker A and B motifs [109], that form the helicase or DEAD motor [108]. DEAD is the acronym for the conserved sequence—Asp-Glu-Ala- C-terminal domain Asp—in this family of proteins. The glutamate is the catalytic base that activates water for hydrolytic attack on ATP [110] and the aspartate li- gates the Mg2+ cofactor of ATP [109,111]. In SecA the two subdomains are called nucleotide-binding domain (NBD) (Chatzi et al., in press, this WD volume) (Fig. 4) and IRA2 (intramolecular regulator of ATPase2). The ATPase active site in SecA is located at the interface of the two PBD subdomains [112]. Recent biophysical data [111] show that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. SecA also has two specificity domains, which distinguishes it from other SF2 proteins: the C-domain that is linked to IRA2 and the SD preprotein binding domain (PBD) which is rooted inside NBD. The C-domain contains 4 substructures: the long α-helical scaffold domain (SD); the conserved helix-loop-helix IRA1 switch (also known as two-helix finger); the α-helical wing domain (WD); and the C-terminal tail (C-tail) that binds Zn2+ [105,108]. The SD con- NBD tacts all other domains of SecA, controls the opening and closing of the DEAD motor, and is involved in preprotein binding along with the PBD [113]; IRA1 negatively regulates cytosolic SecA ATPase ac- IRA2 tivity (in the resting state) [114], makes important interactions with the SecYEG channel (during translocation) [115] and contains residues involved in SecA dimerization [116,117];WDandtheC- tail are involved in SecB and lipid binding [113]. The PBD contains an anti-parallel β-strand (stem) and a bilobate ADP globular domain (bulb). The crystal structures show that the PBD bulb undergoes an approximately 75° swiveling motion between a ‘closed’ Fig. 4. Crystal structure of B. subtilis SecA. Ribbon diagram of B. subtilis SecA showing the and an ‘open’ state (which could accommodate preproteins or regions structural domains: NBD in yellow, IRA2 in blue, the PBD in orange, the SD in green, and of SecYEG) [108]. The movement of PBD creates two grooves on the the WD in cyan. ADP is shown in a ball-and-stick representation. surface of SecA: the large groove 1, formed between PBD, WD and SD Figure reproduced from [101] with Copyright (2004) National Academy of Sciences, U.S.A. S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1773 which exists in a compact, inactive, ADP-bound state (quiescent, stage We confine our discussion to SecA (or SecA1) whose essentiality is 1), binds with high affinity to SecYEG leading to a conformational confirmed in several bacteria including pathogens such as H. influenzae change. This results in a moderate increase in ATPase activity—termed [135], mycobacteria [136], S. aureus [137] and Burkholderia the membrane ATPase—and an increased affinity for preproteins and pseudomallaei [138]. SecA is a broad-spectrum, druggable target SecB (priming, stage 2). SecB targets the unfolded preprotein to SecA [130] for which structural, biochemical and biophysical information, bound to SecYEG. Preprotein binding to SecA results in a substantial high-throughput screening assays and inhibitors are progressively increase in ATPase activity—termed the translocation ATPase—due to evolving. the opening of a highly conserved salt bridge (gate 1) at the base of the DEAD motor which renders it in a loose state with decreased 3.1.3. Druggable sites affinity for ADP (triggering, stage 3). The release of ADP changes the SecA ATPase activity is essential for translocation [26] while SecA conformation of SecA PBD together with bound preprotein. SecB is re- helicase activity is not essential for translocation in vivo. An obvious leased (pre-insertion, stage 4). ATP binding to the DEAD motor drives strategy is to find inhibitors of SecA ATPase activity. However, the nucle- additional conformational changes that enables deeper insertion of otide binding (DEAD motor) domain of SecA is conserved in other SecA into the membrane. The bound signal peptide penetrates the ATPases as well (e.g., ATP-dependent helicases, F1 ATPase, AAA ATPases membrane, 20–30 amino acid segment of the mature part is threaded and ABC transporters [111]). Consequently catalytic inhibitors of SecA into the SecYEG channel (ATP stroke, stage 5). ATP hydrolysis causes a are likely to cross-react with other ATPases. This problem was encoun- partial release of preprotein into the channel and conformational tered during early high-throughput screens [26]. Although catalytic changes in SecA. This allows SecA to revert back to its compact, ADP- inhibitors can be optimized by chemical modification, allosteric inhibi- bound state and to de-insert from the membrane, a step accelerated tors that disrupt SecA ATPase activity by targeting regions other than by the proton motive force (de-insertion, stage 6). The next round of those present in human ATPases offers better prospects [26]. In fact, ATP binding and hydrolysis proceeds as SecA PBD moves further most SecA inhibitors identified (Section 3.3) either compete with ATP down the preprotein, triggering ADP release. The steps 3 to 6 are repeat- binding or prevent ATP hydrolysis by binding to an allosteric site on SecA. ed till the protein is translocated across the membrane (multiple rounds An alternate approach is to identify molecules that prevent SecA- of catalysis, step 7). ligand interactions, resulting in the inhibition of protein translocation. Recently, SecA has also been proposed to function alone as an ion This area is currently unexplored and there are no inhibition assays spe- and protein-conducting channel [124,125]. cifically designed for this purpose. Two important interactions that could be targeted are SecA-SecYEG and SecA-preprotein interactions. 3.1.2. Relevance as antibiotic target The structures of SecA bound to nucleotide, SecYEG [103] a signal pep- The factors that make SecA an attractive target are: tide and a tripeptide [104,105,119] as well as the increasing knowledge of the binding sites on SecA for interaction with these ligands [116,139], (i) SecA is ubiquitous and conserved in bacteria [25]. provide the basis for rational inhibitor design. Several pockets at the (ii) It is essential for bacterial cell viability as demonstrated by con- SecA surface are proposed as druggable sites in addition to the large structing temperature-sensitive mutants of E. coli and B. subtilis groove 1 and a deep pocket formed by residues of the PBD and the C- [126–128] and by gene knockout technique in E. coli [129].Growth terminal domain of SecA [130]. This is a unique approach as there are of temperature-sensitive secA mutants at non-permissive temper- currently no drugs based on this type of interaction. atures (42 °C) leads to defective protein secretion and cell division and finally to cell death [127,128]. 3.2. SecA inhibition assays (iii) SecA or a homolog is absent from humans as confirmed by sub- tractive genomics approach [130]. 3.2.1. In vitro assays (iv) SecA is a cytoplasmic target and a peripheral membrane protein. Although any potential inhibitor needs to cross the membrane 3.2.1.1. ATPase activity assay. ATPase activity of SecA alone (basal/ barrier(s), SecA should still be accessible to many compounds. intrinsic) or SecA with membrane (membrane ATPase) or SecA with During some stages of the catalytic cycle SecA is expected to membrane and preprotein (translocation ATPase), is commonly mea- have parts buried in the membrane [131–133].Therecentview sured by colorimetric determination of the released phosphate using of SecA functioning as an ion and protein conducting channel malachite green [140]. The SecA translocation ATPase assay, adapted [124] also means that SecA might be more accessible for potential to a 96-well format by researchers at Pfizer [141], demonstrated the inhibitors. activity of SecA inhibitor, CJ-21,058 (Section 3.3.2). To simplify the (v) SecA has several interactions and enzymatic activities, providing in vitro ATPase assay for high-throughput inhibitor screening, a SecA greater options for targeting. mutant (Trp to Ala, at position 775, E. coli numbering) was designed (vi) Targeting SecA will drastically reduce bacterial virulence since the with elevated intrinsic ATPase activity that supports normal protein newly synthesized virulence proteins that depend on SecA- translocation both in vitro and in vivo [142]. The use of the SecA mutant mediated protein transport will be locked up in an unfolded increases the sensitivity of the assay compared to the native SecA which state within the bacterial membrane. has a low basal ATPase activity and in addition, obviates the need for Mycobacteria and some Gram-positive bacteria (including Listeria, membrane (inner membrane vesicles or proteoliposomes containing Staphylococcus and some Streptococcus species) possess two homo- SecYEG) and unfolded preprotein components in the reaction mixture. logues of SecA called SecA1 and SecA2 (see review [134]). In bacteria The assay standardized in a 384-well format is robust (average Z′ factor with two SecAs, the two enzymes have non-overlapping functions: [69] of 0.89), and led to the identification of novel 5-amino-thiazolo SecA1 is the essential ‘house-keeping’ ATPase in the Sec-pathway [4,5-d] pyrimidine inhibitors [143] (Section 3.3.2). Another screening (similar to SecA of E. coli and B. subtilis) while SecA2 is a nonessential assay utilized a truncated E. coli SecA devoid of the C-terminal regulato- ATPase, responsible for exporting a subset of proteins which are linked ry domain—responsible for the low intrinsic ATPase activity—to identify to virulence in many pathogens. Exceptions are C. glutamicum (non- fluorescein analogs as inhibitors [144]. The latter two screening assays pathogenic soil bacterium) and C. difficile (the major cause of nosocomi- are designed to identify active site inhibitors. al antibiotic-associated diarrhea), in which SecA2 is also essential [134]. The conservation of the functional domains across SecA (and SecA1) 3.2.1.2. Preprotein translocation assay. SecA-mediated translocation and SecA2, offers the possibility of developing inhibitors targeting of unfolded preprotein substrates (e.g. alkaline phosphatase) in vitro both SecA proteins to inhibit growth as well as virulence [24]. is monitored by SDS-PAGE followed by immunodetection [140]. 1774 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Another gel-based assay involves 35S-labeled proOmpA as a marker, a hydrophilic tetramic acid moiety and a hydrophobic bicyclic moiety which is quantified by densitometry [144,145]. These assays are low- [141]. CJ-21,058 (Table 5, compound 2) inhibits translocation ATPase throughput and used as secondary assays to confirm SecA inhibition. activity of E. coli SecA (IC50 of 15 μg/ml), as demonstrated in an in vitro assay developed by Pfizer (see Section 3.2.1.1). The inhibitor is 3.2.2. In vivo assays (target-based) antibacterial against multi-drug resistant S. aureus and Enterococcus faecalis (MIC 5 μg/ml) while it has no activity against E. coli and Strepto- 3.2.2.1. Whole cell reporter assay. A cell-based assay involving a recombi- coccus pyogenes. The correlation between SecA inhibition and antibacte- nant E. coli strain with a SecA-LacZ fusion reporter system was developed rial activity and the effect of CJ-21,058 on the intrinsic ATPase activity of by Wyeth-Ayerst Research for screening for SecA inhibitors [146].The SecA was not reported [141]. Docking of CJ-21,058 into the active site of assay is based on the property of SecA to autogenously regulate its E. coli SecA shows that the molecule binds at the ATP binding site, indi- mRNA translation in response to changes in secretion levels. Under cating that it probably competes with ATP for binding [144]. normal secretion conditions SecA binds to its own mRNA, inhibiting We undertook a high-throughput screen of ~27,000 diverse small- translation but when secretion is blocked, mRNA is released and transla- molecules against E. coli SecA mutant with elevated intrinsic ATPase action is upregulated. One drawback is that the assay appears to preferen- tivity (Section 3.2.1.1) [142]. This led to identifying several inhibitors tially select compounds that affect membrane integrity [146]. belonging to different chemical classes, including nipecotic acid deriva-

tives and pyrrolopyrimidines with IC50 values ranging from 50 to 150 3.2.2.2. Reduced expression of SecA. An assay developed at Merck [147] μM against wild-type E. coli SecA [130,142]. A further screen of a library utilizes an S. aureus strain that generates antisense RNA against secA of synthesized thiazolo[4,5-d]pyrimidine derivatives, identified several thereby rendering the strain hypersensitive to inhibitors of SecA. A ATPase inhibitors of E. coli and S. aureus SecA1 [143].Kineticanalysisof two-plate agar-based differential sensitivity screen—comparing the ef- the most potent inhibitor (Table 5,compound3) revealed a mixed-type fect of compounds on the sensitized strain versus the wild-type con- inhibition, with an inhibition constant of 60 μM against translocation trol—conducted on a high-throughput scale led to the identification of ATPase activity of S. aureus SecA1 [130]. The antibacterial activity was the natural product inhibitor, pannomycin [147] (Section 3.3.3). not significant (unpublished data). Fluorescein analogs (hydroxanthenes), Rose Bengal and Erythrosin 3.2.2.3. A common screen for Sec-pathway inhibitors. A high throughput B(Table 5, compounds 4 and 5), identified from an in vitro screen screening assay designed by Cowther et al. [148] utilizes a recombinant employing truncated unregulated E. coli SecA (Section 3.2.1.1), proved

E. coli strain that accumulates β-galactosidase (β-gal) in its cytoplasm if effective inhibitors with IC50 of 0.5 and 2 μM, respectively [144]. translocation through SecYEG is blocked. Presumably, toxic compounds Rose Bengal is relatively more potent than Erythrosin B, also against and nonspeciﬁc protein synthesis inhibitors prevent β-gal production the full-length (regulated) SecA from E. coli and B. subtilis, as seen in and do not show up as hits. Although, an initial screen of 800 com- Table 5. Rose Bengal strongly inhibits the preprotein translocation pounds did not yield reproducible hits, the assay is reliable (average Z′ in vitro with IC50 of about 0.25 μM (the highest potency among the factor of 0.60, manual screen) and suited for 384-well format. SecA inhibitors). While the inhibitor lacks antibacterial activity against the wild-type E. coli, it has equally potent antibacterial activity against 3.3. Overview of SecA inhibitors a permeable leaky E. coli mutant (NR698) and wild-type B. subtilis

(IC50 of 3.1 μM). Thus, the fluorescein analogs are more effective on The different approaches (in vitro, in vivo and in silico) for finding Gram-positive bacteria for which outer membrane permeability is not SecA inhibitors have resulted in only a few molecules, described below. aproblem[144]. It is interesting to note that unlike sodium azide, Rose Bengal and Erythrosin B inhibit all three ATPase activities of SecA 3.3.1. Discovery of sodium azide, a research tool for SecA inhibition studies (basal, membrane and translocation), albeit with different efficiencies Sodium azide was the first SecA inhibitor reported (in 1990s), as observed in Table 5. An in silico modeling study shows that the fluo- although its antibacterial activity was known since the late 19th century rescein inhibitors bind at the high affinity ATP-binding site on SecA and [130]. Sodium azide inhibits translocation ATPase activity (but not that Rose Bengal and the SecA inhibitor, CJ-21,058 occupy the same intrinsic ATPase activity) of SecA [Table 5, compound 1], probably by position with the same orientations [144]. Huang et al. [144] indicate blocking the de-insertion step and trapping SecA in the membrane that Rose Bengal and Erythrosin B are competitive inhibitors against [132,149]. Sodium azide also inhibits other ATPases including mito- ATP and are likely to be general ATPase inhibitors which are more effec- chondrial F-ATPase [150] and several enzymes including cytochrome c tive on the catalytic SecA ATPase. oxidase, superoxide dismutase and alcohol dehydrogenase [151] and A recent SAR study indicates that the xanthene ring in Rose Bengal is is unsuitable as an antibiotic. Nevertheless, the azide inhibitor demon- essential for activity and that simplified analogs with half the molecular strated SecA as an antibacterial target and has served as a positive con- weight of Rose Bengal can be as potent as the parent compound [152]. trol in SecA inhibition assays [141,142,146,148]. The exact mechanism by which sodium azide works as a SecA inhibitor is not known. Based on the mechanism of inhibition of the mito- 3.3.3. Discovery of Pannomycin by the in vivo approach chondrial F-ATPase by sodium azide, a comparison can be drawn Pannomycin (a substituted cis-Decalin) is a secondary metabolite

[130]. The ternary structure of the complex of F1-ATPase with ADP from the fungus, Geomyces pannorum, identiﬁed in a SecA two-plate dif- and azide [150], shows that the azide anion brings the side chains of ferential sensitivity antisense assay (Section 3.2.2.2) by researchers at two catalytically essential amino acids closer to the nucleotide, creating Merck [147]. Pannomycin was one among over 115,000 natural product a tighter ﬁt with the ADP molecule and stabilizing the ADP-bound state. extracts (derived from both actinomycetes and fungi) screened in the

Since SecA and F1-ATPase bind to adenine nucleotides with a very sim- high-throughput antisense assay, which had a primary hit rate of 0.1%. ilar geometry [105], a comparable mechanism could explain how sodi- Pannomycin [Table 5, compound 6] is structurally similar to the SecA in- um azide stabilizes the membrane-inserted, ATP-bound state of SecA hibitor CJ-21,058, which is comprised of decalin linked to a tetramic [131,132]. acid. Pannomycin has a weak antibacterial activity (in mM range) against Gram-positive S. aureus Smith strain, E. faecalis and B. subtilis 3.3.2. Discovery of equisetin (CJ-21,058), thiazolo[4,5-d]pyrimidine and as shown in Table 5,butnotagainstS. pneumoniae, H. influenzae, E. coli fluorescein analogs by the in vitro approach and Candida albicans. Parish et al. [147] attribute the higher antibacterial The first natural product inhibitor of SecA, CJ-21,058, isolated from activity of CJ-21,058 to the presence of tetramic acid and suggest further an unidentified fungus, is an analog of Fusarium toxin equisetin, with optimization of the pannomycin decalin scaffold. However, the SecA S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1775

Table 5 Inhibitors of SecA with their in vitro and in vivo activities.

Compound IC50 MIC against micro-organism Ref.

a 1 Sodium azide (NaN3)5mMND [193] 2 38.7 μMa 12.9 μM S. aureus 01A1105 [141] 12.9 μM E. faecalis 03A1069

3 5-amino-thiazolo[4,5-d]pyrimidine ~200 μM a 135 μM b ND [143]

4 Rose Bengal 0.9 μM a N1mM E. coli MC4100 [144] 25 μM b 3.1 μM E. coli NR698 5 μM c 3.1 μM B. subtilis 168 7 μM d 0.25 μM e

5ErythrosinB 10 μM a N10 mM E. coli MC4100 [144] 21 μM b 250–500 μM E. coli NR698 12 μM c 250–500 μM B. subtilis 168 70 μM d 4 μM e

6 Pannomycin ND 0.4 mM B. subtilis [145] 1.4 mM S. aureus Smith 1.4 mM E. faecalis

7 ND 3.2 μM S. aureus MN8 [146]

8 SEW-05929 ~100 μM f ND [151,154]

9 HTS-12302 ~100 μM f ND [151,154]

(continued on next page) 1776 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Table 5 (continued)

Compound IC50 MIC against micro-organism Ref. 20 μM b N50 μM E. coli MC4100 [154] 2 μM f N50 μM E. coli NR698

11 50 μM b N50 μM E. coli MC4100 [154] 2 μM f N50 μM E. coli NR698

12 60 μM b N50 μM E. coli MC4100 [154] 20 μM E. coli NR698

13 2.5 μM g ND [155]

14 0.25 μM g 759 μM h A. tumefaciens [156]

15 0.92 μM g 586 μM h A. tumefaciens [156]

16 0.48 μM g 609 μM h A. tumefaciens [156]

17 0.64 μM g 333 μM h A. tumefaciens [156]

18 0.44 μM g 639 μM h A. tumefaciens [156] S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1777 inhibitory activity of pannomycin has not been further validated and its were found to inhibit the intrinsic ATPase activity of Ca. L. asiaticus SecA mode of action remains unclear. at nanomolar concentrations (IC50, 0.25–0.92 μM). The inhibitors show The antisense screening assay is highly sensitive which is reflected antimicrobial activity against Agrobacterium tumefaciens, which is phy- by the fact that it could identify a weak inhibitor such as pannomycin. logenetically related to Ca. L. asiaticus, although at a high concentration An earlier attempt at high-throughput screening using a SecA-LacZ re- (see Table 5). While there is a potential to optimize these molecules to- porter fusion system (Section 3.2.2.1) identified a few molecules that in- wards developing antimicrobial agents against Ca. L. asiaticus, it would duced SecA expression (which occurs when secretion is blocked) but all also be interesting to know their spectrum of activity against other bac- the hits were found to have a deleterious effect on the membrane [146]. terial pathogens. The most potent hit is shown in Table 5 (compound 7). Recently, a 3D-Quantitative Structure–Activity Relationship (3D- QSAR) model was developed using known SecA inhibitors [157].As the numbers of SecA inhibitors increase, such models could facilitate 3.3.4. Discovery of inhibitors of SecA (of E. coli and Candidatus Liberibacter virtual screening and compound optimization. asiaticus) by an in silico approach All three approaches used for SecA inhibitor screening have been The availability of the crystal structure of E. coli SecA [98], enabled in fruitful in finding effective inhibitors. In the case of SecA, as many effec- silico screening of compounds against SecA. Structure-based virtual tive inhibitors (in vitro) lack antibacterial activity especially against screening involves docking of compounds (from an entire library or da- Gram-negative bacteria, it is tempting to infer that the in vivo approach tabase) into the active site or binding pocket and estimating theoretical holds greater promise in finding novel antibacterials. binding energies. Based on these, compounds are selected for in vitro testing [151,153]. Screening of 60,000 compounds from a commercial 4. In brief: attempts to target other protein collection (MayBridge database, UK) against E. coli SecA holo-form secretion channels/components (PDB ID: 2FSG), identified 31 hits of which the two best compounds SEW-05929 and HTS-12302 (IC of 100 μMineachcase)[151,154] 50 The three enzymes, SPase II, SPase IV and YidC, the twin-arginine are shown in Table 5 [compounds 8 and 9]. Further optimization led translocation pathway and the Type I to VI secretion systems, are al- to more potent thiouracil derivatives of the parent compound, HTS- ready being evaluated as novel targets. These components/pathways 12302 [154].AsseeninTable 5 [compounds 10, 11 and 12], the thioura- are detailed in other articles in this issue, while this section focuses on cil derivatives inhibit the intrinsic ATPase activity of the full-length their possible use as antibacterial targets. E. coli with IC50 values in the range of 20 to 60 μM. They are highly potent against the truncated E. coli devoid of C-terminal regulatory domain. However, the compounds do not have significant biological activ- 4.1. Targeting the lipoprotein (or Type II) signal peptidase ity against the wild-type E. coli, except compound 12 [Table 5] which has some antibacterial activity against NR698 [154]. SPase II or the lipoprotein signal peptidase (LspA) is responsible for Interestingly, another structure-based virtual screen was directed to- processing lipoproteins (diacylglyceryl modified proteins). The recogni- wards finding SecA inhibitors of (Gram-negative bacterium) Candidatus tion and cleavage site of LspA lies in a region known as the lipobox, Liberibacter asiaticus, the causal agent of Huanglongbing disease of citrus present in the C-region of the signal peptide. The consensus sequence [155]. In this work, a 3D homology model of Ca. L. asiaticus SecA was of the lipobox in B. subtilis and E. coli is L-A/S-A/G-C. Processing by built based on the crystal structure of E. coli SecA (PDB ID: 2FSG) [98]. LspA occurs just before the invariable cysteine residue that is also the A total of 5,016 small-molecules, filtered based on physicochemical site of diacylglyceryl modification [22]. LspA is a potential antibiotic tar- properties from ChemBridge and Specs chemical databases, were used get because: LspA is an unconventional aspartic acid protease which for molecular docking into the active ATP-binding site of Ca. L. asiaticus lacks the conserved Asp-Thr/Ser-Gly motif present in (eukaryotic and SecA. Among the 20 hits selected for biological activity studies, viral) aspartic proteases [158]. LspA is absent in eukaryotic cells [14],es- the highest potency (IC50 of 2.5 μMagainstCa. L. asiaticus SecA) was ob- sential for viability in Gram-negative bacteria such as E. coli but not in served for compound 13, Table 5 [155]. The antimicrobial activity is not Gram-positive bacteria such as B. subtilis [158] or Lactococcus lactis known. [159]. However, in pathogens including M. tuberculosis, LspA plays an Wang et al. [156], further optimized the homology model of Ca. important role in processing virulence determinants. An LspA mutant L. asiaticus SecA using three PDB structures (2VDA, 2FSF and 2FSG) as in (Gram-positive, acid-fast) M. tuberculosis, is severely attenuated in templates and virtually docked 20000 compounds from the com- virulence tuberculosis models [160]. The active site is accessible to ex- mercially available ZINC database. Twenty compounds were selected ogenously added potential inhibitors. In B. subtilis LspA, the catalytic res- based on their predicted binding to SecA and tested in vitro for their in- idues are placed on the extracytoplasmic side, just below the membrane hibitory activity. Five of these molecules (Table 5,compounds14 to 18) surface [158].

Notes to Table 5: NR698 is a permeable leaky mutant of E. coli. ND: not determined. compound 2, (E)-1-benzyl-3-[(1-{4-[4-(4-{1-[(E)-[(N-benzylcarbamimidoyl)imino]amino]ethyl}phenyl)piperazin-1-yl]phenyl}ethyl)imino]guanidine; compound 10,4-(4- bromophenyl)-2-({[4-({[4-(4-bromophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl]sulfanyl}methyl)phenyl]methyl}sulfanyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile; compound 11, 2-({[4-({[5-cyano-6-oxo-4-(4-phenylphenyl)-1,6-dihydropyrimidin-2-yl]sulfanyl}methyl)phenyl]methyl}sulfanyl)-6-oxo-4-(4-phenylphenyl)-1,6-dihydropyrimidine-5- carbonitrile; compound 12, 2-(benzylsulfanyl)-6-oxo-4-(4-phenylphenyl)-1,6-dihydropyrimidine-5-carbonitrile; compound 13, 2-(1H-1,3-benzodiazol-2-ylsulfanyl)-N-(2- {[(cyclopropylcarbamoyl)methyl]sulfanyl}-1,3-benzothiazol-6-yl)acetamide; compound 14, N-(5-ethyl-1,3,4-thiadiazol-2-yl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide; compound 15, 3-amino-6-phenyl-4-(3,4,5-trimethoxyphenyl)thieno[2,3-b]pyridine-2-carboxylic acid; compound 16, 4-[(Z)-N,N′-diaminoimidamido]-N-{1-[(4-methoxyphenyl) carbamoyl]cyclohexyl}benzamide; compound 17, N-(6-methoxy-1,3-benzothiazol-2-yl)-2-[(5-methyl-1H-1,3-benzodiazol-2-yl)sulfanyl]acetamide; compound 18, (10R)-5-benzoyl- 12-oxo-10-(thiophen-2-yl)-2,9-diazatricyclo[9.4.0.03,8]pentadeca-1(11),3,5,7-tetraen-9-ium. a IC50 against translocation ATPase activity of E. coli SecA. b IC50 against intrinsic ATPase activity of E. coli SecA. c IC50 against membrane ATPase activity of E. coli SecA. d IC50 against intrinsic ATPase activity of B. subtilis SecA. e IC50 against in vitro translocation of E. coli preproteins. f IC50 against intrinsic ATPase activity of truncated, unregulated E. coli SecA (EcN68 SecA). g IC50 against intrinsic ATPase activity of Ca. L. asiaticus SecA. h The MBC value is shown instead of the MIC value. 1778 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

Although LspA has not been characterized in great detail and no D(T/S)G motif and the pH optimum for in vitro activity is near neutral high-throughput assays are reported yet, at least two effective natural as opposed to acidic (pH 2–4) [167]. The active site of the enzyme is ac- product inhibitors (Table 6, row 1) are known to block its activity. cessible to inhibitors [167]. Globomycin, a cyclic peptide antibiotic [161] and its synthetic deriva- Resistant to the classical protease inhibitors (including pepstatin, a tives [162] inhibit SPase II in a non-competitive manner and are bacte- general aspartic acid inhibitor), Type IV signal peptidase is inhibited by a ricidal against enteric Gram-negative bacteria e.g., E. coli. Recently, combination of EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Myxovirescin/Megovalicin/M-230B, a macrocyclic metabolite (polyke- hydrochloride) and glycinamide (known to inhibit non-pepsin-like acid tide) produced by myxobacteria, was shown to target LspA with 2 to proteases) [167]. Rationally designed peptides, mimicking the cleavage 10 fold better whole cell activity (E. coli MG1655, MIC = 4 μg/ml) com- site region of its native substrate inhibit the cleavage activity of Type IV pared to Globomycin (E. coli MG1655, MIC = 8 μg/ml) [163].Ithas signal peptidase in vitro [170]. Chemical inhibitors of SPase IV, identiﬁed broad-spectrum antibacterial activity and is non-toxic to eukaryotic by in vitro screening include Voltaren (IC50 ~110μM) and γ-secretase cells, including human cells. Synthesis of Globomycin [164] as well as inhibitor VI (IC50 ~500μM) (Table 6, row 2), among others [170]. Myxovirescin A1 [165,166], has paved the way for SAR studies. The Search for potent inhibitors, a detailed understanding of the basic questions that remain to be addressed are: could these inhibitors characteristics of the enzyme, structure, and distribution (pres- lead to effective drugs? What is the structure of the active site? How dif- ence/absence in different bacterial species) will help in further ferent is the structure of LspA in Gram-positive and Gram-negative bac- evaluating SPase IV as a target. teria? Why is the enzyme essential in some bacteria, but not in others? 4.3. Targeting YidC (Sec-dependent or independent) membrane protein 4.2. Targeting the Type IV prepilin peptidase (Type IV signal peptidase) insertion

Type IV signal peptidases—identiﬁed in several Gram-negative bac- In the case of membrane proteins, the hydrophobic transmembrane teria and an increasing number of Gram-positive bacteria—play an segment region in the nascent protein serves as the signal for targeting essential role in processing the precursors of Type IV pilin (surface and insertion into the membrane. These proteins are routed to the structures often involved in virulence) and prepilin-like proteins (secre- SecYEG translocase by Signal Recognition Particle (SRP)-mediated co- tory proteins which possess a Type IV signal peptide, but are not pilin translational targeting [171]. Presumably, a lateral gate in the SecYEG subunits) [167]. The Type IV SPases span the membrane eight times, channel allows membrane protein insertion with the help of YidC have their active site residues close to the membrane boundaries and which is positioned close to the channel gate [172]. YidC is an integral cleave their substrates within the cytoplasm, just proximal to the mem- membrane protein that assists in the lateral insertion and assembly of brane surface [22]. The substrate recognition and cleavage site of the membrane proteins into the lipid bilayer via the Sec-translocase, but SPase IV differ from that of SPases I and II. SPase IV cleaves between can also independently function as an insertase (see review [173]). the residues Gly and Phe in a conserved region located between the N YidC is essential for cell viability [174]. While Gram-negative bacteria and H regions of the signal peptide. It is a bifunctional enzyme, also re- have one YidC, most Gram-positive bacteria have two YidC homologs sponsible for N-methylation of the Phe at position +1 relative to the (YidC1 and YidC2) [171].Deletionofbothhomologswasshowntobele- cleavage site [22]. thal in B. subtilis [175]. YidC homologs are also present in mitochondria Type IV SPase is considered an antivirulence target. The enzyme (Oxa1 and Oxa2) and chloroplasts (Alb3) but absent in ER and plasma plays a central role not only in Type IV pilus biogenesis but also in membrane of eukaryotes [176]. The YidC/Oxa/Alb3 members share con- toxin and other enzyme secretion, natural competence, DNA transfer served hydrophobic regions comprising 5 transmembrane domains, and bioﬁlm formation [168]. Marsh and Taylor [169] demonstrated representing the catalytically active part [171]. Partial functional com- that mutation of the V. cholerae SPase IV gene attenuates virulence plementation has been demonstrated between the homologs Oxa 1 in vivo. SPase IV does not have eukaryotic counterpart. It is an unusual and YidC 2 [176] as well as between Oxa2 and YidC1/YidC [177,178].Al- aspartic acid protease which differs from the majority of aspartic acid though the YidC/Oxa/Alb3 family has a common ancestry, the homologs proteases in that the active site aspartic acids are not found in the in bacteria and mitochondria have evolved independently [176].Given

Table 6 Efforts in targeting other protein secretion components or pathways for ﬁnding of novel antibacterial targets.

Sl. no. Component/pathway Screening assay Inhibitor(s) Reference

1. Lipoprotein signal peptidase -NA- Globomycin and Myxiviriscin [161–163] (LspA) or the Type II SPase 2. Type IV prepilin peptidase In vitro preprotein processing assay Voltaren and γ-secretase inhibitor VI [170] or the Type IV SPase 3. YidC E. coli strain underexpressing yidC gene Eugenol and Carvacrol [179] 4. Twin-arginine translocation (Tat) Primary screen measures the extracellular activity of N-phenyl maleimide (one of the two potent [186] phospholipase C (a potent toxin and a Tat-dependent inhibitors identiﬁed) secretion product) in P. aeruginosa. Several follow-up assays to evaluate Tat-functionality in vivo 5. Type II secretion system The primary bioluminiscent reporter screen uses a 5,7-dichlorohydroxyquinoline, one among [194] P. aeruginosa strain responsive to SecA the 9 inhibitors effective against P. aeruginosa depletion or inhibition. Secondary assays (β-lactamase, and Burkholderia pseudomallei phospholipase C and elastase secretion) identify Sec/Tat/Type II inhibitors. 6. Type III secretion system Luciferase reporter gene assay in Yersinia pseudotuberculosis Salicylidene acylhydrazides, effective against [190,195] Y. pseudotuberculosis with broad-spectrum activity against Gram-negative bacteria (e.g., Chlamydia, Shigella, and Salmonella species) 7. Type IV secretion system In vivo assay that measures interaction of Brucella VirB8 Inhibitor (B8I-2), belongs to salicylidene acylhydrazide [196,197] (an inner membrane protein) with itself and other class of molecules, active against Brucella abortus. components of the Type IV secretion apparatus. VirB8 is an essential and conserved component of the Type IV secretion system. S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783 1779 the homology, whether YidC can be selectively inhibited without any resulted in high-throughput screening and identiﬁcation of inhibitors harm to human cells, needs further investigation. of the secretion systems (Table 6, rows 5 to 7). We refer the interested A recent study utilized an antisense RNA-mediated, yidC down- reader to [190–192] for detailed reviews on the targeting of secretion regulated E. coli strain to identify two antibacterial essential oils (euge- systems for bacterial virulence inhibitors. It must be noted that the com- nol and carvacrol) that possibly targets YidC [179] (Table 6, row 3). The ponents that make up the Type I to VI secretion apparatus are inserted mode of action of the molecules is not known. Currently, no other inhib- into the membrane or exported by the Sec-system [22],andhence, itors or assays are reported for this target. In addition, knowing the targeting the Sec-pathway would also render these secretion systems exact molecular mechanism by which YidC operates or interacts, a sub- defunct. ject of ongoing research [174], will contribute greatly to this line of The alternative approach of using antivirulence targets to disarm work. bacteria and contain infection also looks promising. However, the challenges of exploiting them in monotherapy or combination therapy will 4.4. Targeting the twin-arginine translocation pathway only become clear once the inhibitors are developed into drugs.

The Tat-pathway, which uniquely transports folded proteins across 5. Concluding remarks the cytoplasmic membrane, operates in many bacteria. The Tat-pathway gets its name from two highly conserved arginine residues in the signal The Sec-pathway components, SPases I and SecA are universal, con- peptide region of the protein, recognized by the Tat-apparatus for mem- served and essential in bacteria and can be differentially targeted with- brane targeting and transport. The Tat-system is comprised of either out significant harm to humans or animals. They are characterized and three or two essential integral membrane components, TatABC (as in validated targets in non-pathogenic as well as pathogenic bacteria in- Gram-negative E. coli) or TatAC (as in Gram-positive Bacillus), respec- cluding E. coli, S. aureus, P. aeruginosa and M. tuberculosis. Different ap- tively [180,181]. The in vitro data suggests that the subsequent steps proaches have been applied to discover SPase I and SecA inhibitors, for in Tat-mediated transport in E. coli are: binding of the signal peptide re- over two decades. We have witnessed a transition in the approach gion of the preprotein to TatC which forms a complex with TatB, trans- from random high-throughput screening using the isolated enzyme to location of the protein through a channel of varying size formed by TatA whole cell antibacterial or target-based whole cell screening. SPase I in- oligomers and release of the mature protein after removal of SP by the hibitors, arylomycins (including actinocarbasin) and krisynomycin are SPase I. The translocated proteins are subsequently released into good starting points for optimization towards novel antibacterials. the extracellular environment (in Gram-positive bacteria) or into SPase I, being a single-enzyme target, can evolve resistance by point- the periplasm (in Gram-negative bacteria) for further transport across mutation. Preclinical assessment of resistance development is impor- the outer membrane and, where applicable, outside the cell via tant while evaluating new drug leads based on SPase I. SPases I can the Type II secretion system [182]. The Tat-pathway is identified as also be exploited in combination therapy as demonstrated by synergy an antivirulence target because several bacterial pathogens (e.g., of SPase I inhibitors with β-lactams and an aminoglycoside, gentamicin. M. tuberculosis, L. pneumophila, P. aeruginosa and B. pseudomallei) utilize This idea is further supported by the observation that the SPase I the Tat-pathway for transporting extracellular enzymes and other viru- inhibitor, M131, in combination with Imipenem suppresses emergence lence determinants essential for pathogenesis [183]. The Tat-system is of resistance. There are currently no antibacterial candidates targeting also functional in plants but no homologues are seen in animals. In SecA. Although a few effective active site inhibitors have been identified P. aeruginosa,atatC mutant is severely attenuated in a rat pulmonary using in silico, in vitro and in vivo approaches, allosteric inhibitors with infection model [184],whileinM. tuberculosis, the Tat-pathway is good antibacterial properties are desirable. Assays specifically designed seemingly essential for growth, at least under standard laboratory for finding SecA inhibitors that block its interaction with SecYEG and or conditions [185]. preproteins might be beneficial. Despite several crystal structures of Recently, a high-throughput assay developed for screening inhibi- SecA and continuing efforts, its mode of action is not completely under- tors of the Tat-pathway identified two compounds that directly affect stood. The availability of high-resolution crystal structures especially its function [186] (Table 6, row 4). The current limitation of the Tat- that of SecA actively engaged in translocation of preprotein in the pathway as a novel target is the lack of detailed structural and functional protein conducting channel could fine tune the in silico approach for data across different bacterial species due to the relatively recent history screening or optimizing the identified molecules. Similarly, crystal of its discovery (in the mid-nineties). Moreover the Tat-pathway is structure of the full length SPase I including that from Gram-positive present in some but not all bacteria, which narrows down its spectrum. bacteria will be helpful. The Tat-pathway and the bacterial secretion The Tat-pathway is dependent on the Type I SPase for processing systems—Type II, Type III and Type IV—have been pursued as anti- and release of its preproteins from the membrane [187]. Thus targeting virulence targets, resulting in a few inhibitors. In comparison with SPase I will also disrupt the Tat-mediated protein secretion. SPase I or SecA, these targets are not necessarily broad-spectrum and are not bactericidal. In addition, targeting the SPase I will disrupt the 4.5. Targeting the Type I to VI secretion systems in Gram-negative bacteria Tat-pathway and the terminal branches of the Sec-pathway (Type II and Type V secretion systems), while targeting SecA will either disrupt Protein secretion in Gram-negative bacteria is either a one-step or or severely affect the additional (Type I to Type VI) secretion systems. two-step process. In the latter case, the proteins first cross the inner The Sec-pathway components, YidC, SPase II and SPase IV are also membrane (via the Sec-pathway or in some cases, the Tat-pathway) valid targets which need further work. In essence, the Sec-pathway and then cross the outer membrane to reach the external environment. does offer some good targets for novel antibacterial research. Gram-negative bacteria possess at least six specialized secretion sys- Supplementary data to this article can be found online at http://dx. tems (Type I to VI), of which Type II and Type V are terminal branches doi.org/10.1016/j.bbamcr.2014.02.004. of the Sec-pathway while the others export proteins directly from the cytoplasm to the outside milieu or even into the eukaryotic host cells Acknowledgements (see review [188]). These secretion systems are required for transport of proteins essential for pathogenesis (e.g., toxins, enzymes, anti-host Part of the experimental work described in this overview was car- factors), biofilm formation, and interaction with host or nutrient acqui- ried out in the framework of Grant SBO 50164 from IWT (Institute for sition, and hence, are good anti-virulence targets [189], but are not es- the Promotion of Innovation by Science and Technology in Flanders). sential for viability. Components of the Type II, Type III and Type IV Financial support is gratefully acknowledged. We thank two anony- secretion systems are already pursued as targets and this line of work mous reviewers for their helpful suggestions. 1780 S. Rao C.V. et al. / Biochimica et Biophysica Acta 1843 (2014) 1762–1783

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