DOI: 10.1002/cbic.201402358

A Simple Fragment of Cyclic Acyldepsipeptides IsNecessary and Sufficient for ClpP Activation andAntibacterial ActivityDaniel W. Carney,[a] Corey L. Compton,[a] Karl R. Schmitz,[b] Julia P. Stevens,[a] Robert T. Sauer,[b]

and Jason K. Sello*[a]

The development of new antibacterial agents, particularlythose with unique biological targets, is essential to keep pacewith the inevitable emergence of drug resistance in pathogen-ic bacteria. We identified the minimal structural component ofthe cyclic acyldepsipeptide (ADEP) antibiotics that exhibits an-tibacterial activity. We found that N-acyldifluorophenylalaninefragments function via the same mechanism of action asADEPs, as evidenced by the requirement of ClpP for the frag-ments’ antibacterial activity, the ability of fragments to activateBacillus subtilis ClpP in vitro, and the capacity of an N-acyldi-fluorophenylalanine affinity matrix to capture ClpP from B. sub-tilis cell lysates. N-acyldifluorophenylalanine fragments aremuch simpler in structure than the full ADEPs and are alsohighly amenable to structural diversification. Thus, the stagehas been set for the development of non-peptide activators ofClpP that can be used as antibacterial agents.

Recently, cyclic acyldepsipeptide (ADEP) antibiotics have gar-nered considerable attention because of their potent antibac-terial activity against a broad range of Gram-positive bacterialpathogens and their efficacy in animal models of bacterial in-fection.[1] The cellular target of the ADEPs is ClpP, a componentof the highly conserved Clp proteolytic complexes in bacter-ia.[1c, 2] ClpP is a self-compartmentalized, tetradecameric serinepeptidase that functions in conjunction with accessory AAA+

(ATPases associated with diverse cellular activities) partners,which themselves play important roles in substrate recognitionand regulation of ClpP proteolytic activity.[2d, 3] Clp proteolyticcomplexes degrade a variety of misfolded and native targetproteins, including those involved in regulating stress respons-es and virulence-factor production.[4] ADEPs bind ClpP and dys-regulate its activity by opening its axial pores, yielding a pep-tidase that indiscriminately degrades large peptides and un-structured proteins without the involvement of AAA+ part-

ners, ultimately leading to inhibition of cell division and celldeath.[1c, 2a]

In crystal structures of the Bacillus subtilis ClpP tetradecamerin complex with ADEPs, the small molecules bind with exqui-site specificity at the intra-subunit sites to which AAA+ part-ners, like ClpX and ClpA, also bind.[2b, c, 5] Intriguingly, it hasbeen proposed that the N-acylphenylalanine side chain ap-pended to the ADEP peptidolactone mimics the highly con-served IGF or LGF tripeptide motifs of ClpX and ClpA thatmediate interactions with ClpP.[2b, 5] We find this proposal to bethought-provoking in light of our recent finding that rigidifica-tion of the peptidolactone via incorporation of conformational-ly constrained amino acids can improve ClpP activation asmuch as sevenfold in vitro.[6] These observations motivated usto carry out experiments to define the relative contributions ofthe ADEP peptidolactone and its side chain to ClpP bindingand activation.

For this analysis, we prepared fragments of the ADEP struc-ture (1), including an ADEP peptidolactone with an N-acetylserine residue (2), an N-acetyl-(3,5-difluorophenyl)alanyl pep-tidolactone (3), a serine-prolyl ester coupled to N-(E)-hept-2-enoyl-(3,5-difluorophenyl)alanine (4), and an N-(E)-hept-2-enoyl-(3,5-difluorophenyl)alanine methyl ester (5 ; Scheme 1).The bioactivities of the fragments were assessed in growthinhibition assays by using wild-type B. subtilis AG174, a highlyADEP-susceptible bacterium. Only fragments 4 and 5 exhibitedantibacterial activity, albeit with much lower potency thanintact ADEP (Scheme 1). Clearly, the N-acyldifluorophenylala-nine moiety is necessary and sufficient for antibacterial activity.Interestingly, fragment 4, which has an acyclic portion of theADEP peptidolactone, is less active than fragment 5, which iscomposed of only the N-acyldifluorophenylalanine moiety.

Based on our recent report that conformational flexibility ofthe ADEP peptidolactone strongly influences ClpP binding,[6]

we predicted that substitution of the serine in 4 with eitherallo-threonine (6) or threonine (7, Scheme 2) would yield morerigid and bioactive fragments. However, both compoundsexhibited the same antibacterial activity as fragment 4 (MIC =

32 mg mL�1), suggesting that the allo-threonine and threonineresidues do not significantly or favorably restrict the conforma-tions of the acyclic fragments.

A positional scanning approach was used to define the mini-mal structural requirements for the bioactivity of fragment 5.First, we synthesized analogues with varied acyl chains (8–10 ;Scheme 2). Fragment 8 bears an acyl chain that is common topreviously reported synthetic ADEPs,[1c] and fragment 9 bears

[a] Dr. D. W. Carney, C. L. Compton, J. P. Stevens, Prof. Dr. J. K. SelloDepartment of Chemistry, Brown University324 Brook Street, Providence, RI 02912 (USA)E-mail : jason_sello@brown.edu

[b] Dr. K. R. Schmitz, Prof. Dr. R. T. SauerDepartment of Biology, Massachusetts Institute of Technology77 Massachusetts Avenue, Cambridge, MA 02139 (USA)

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201402358: Experimental procedures for thepreparation of chemical compounds, antibacterial assays, ClpP activationassays, ClpP mutant selection, and chemical proteomics are described inthe Supporting Information.

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the acyl chain of the A54556 ADEP natural products.[1a] Frag-ment 10 is an analogue that we synthesized to test the effectsof acyl group carbon-branching. Fragments 5, 8, and 10 wereequally potent (MIC = 8 mg mL�1), whereas fragment 9 exhibit-ed attenuated activity (MIC = 32 mg mL�1). This result is consis-tent with the literature, wherein analogues with polyunsatura-ted side chains are reportedly less active compared to ana-logues with only a,b-unsaturation.[1d] Fragments 11 and 12(Scheme 2), with saturated acyl groups, had reduced antibacte-rial activity like their ADEP counterparts.[1d] We next studieda pair of analogues in which the difluorophenylalanine wasreplaced by either phenylalanine (13) or leucine (14). The rela-tively modest activity of the fragment with the phenylalanineresidue (13, MIC = 64 mg mL�1) is consistent with the proposalthat the fluorine atoms of difluorophenylalanine are engagedin hydrogen bonds with backbone amides of ClpP.[1d] In con-trast, the analogue containing leucine (14) was completelyinactive. Collectively, the results are consistent with publishedstudies of ADEP structure–activity relationships.[1d]

Next, we explored the effects of several C-terminal manipu-lations on the antibacterial activity of fragment 5. Interestingly,the methyl ester (5) was more potent than either the carboxyl-ic acid or amide analogues thereof (15–18). The low potenciesof the carboxamides were surprising, because the N-acyldi-fluorophenylalanine moiety in the ADEPs is linked to the pep-tidolactone via an amide bond. In any case, three additional

ester analogues (19–21) were prepared. Only the propargylester (21, MIC = 2 mg mL�1) was more potent than 5. Thepotency of the intact ADEPs and the findings that modificationof the C terminus of 5 can improve potency suggest that thestructural context in which the bioactive N-acyldifluorophenyl-alanine moiety is presented is important and can be opti-mized.

We tested our operating assumption that ADEPs and N-acyl-difluorophenylalanine fragments have the same mechanism ofkilling bacteria in a series of genetic and biochemical experi-ments. In initial genetic experiments, an ADEP (1) and frag-ments 4 and 5 were tested for antibacterial activity againsttwo engineered strains of B. subtilis—a spx null strain (AG 1927spx ::neo) and a strain lacking both spx and clpP (AG 1246spx ::neo and clpP ::cat).[7] The clpP-spx null strain was selectedbecause the spx null mutation suppresses the slow growthdefect exhibited by a B. subtilis strain lacking clpP.[7] The spxnull and wild-type strains of B. subtilis, both of which containa functional ClpP, were equally susceptible to each of the threecompounds. Importantly, neither intact ADEP nor fragments 4and 5 were toxic to the spx-clpP null strain (MIC>128 mg mL�1). The requirement of a functional clpP gene forthe toxicity of the fragments indicates that they have the samemechanism as the ADEPs.

We also tested for cross-resistance by selecting for sponta-neously resistant mutants to either 1 or 5 in the spx null strain.Mutants with resistance to the intact ADEP and fragment 5were observed at frequencies of 3 � 10�6 colony-forming units(cfu) and 7 � 10�5 cfu, respectively. As expected, all mutantsresistant to 1 were resistant to 5 and vice versa (MICs>128 mg mL�1). By sequencing the clpP locus in the mutants, wedetermined that resistance was highly correlated with muta-tions in the promoter of the clpP gene or with missense orframe-shift mutations in the clpP open reading frame (see theSupporting Information).

To biochemically validate the proposal that ADEP fragmentsactivate ClpP peptidase activity, they were tested for activationof B. subtilis ClpP in vitro (Figure 1; Figure S2). Fragments wereincubated with B. subtilis ClpP and a fluorogenic decapeptideand initial rates of ClpP-catalyzed decapeptide hydrolysis weremeasured. All fragments exhibited concentration-dependentactivation of ClpP decapeptidase activity and exhibited appar-ent activation constants (Kapp) ranging from 3.9–11 mm. As thebinding affinities fall into a narrow range, the large differencesin bioactivities of the compounds can be primarily attributedto their in vivo stability and/or cell permeability. Nevertheless,the fragment with the most potent antibacterial activity (21)was also the tightest ClpP binder. In any case, the fragments’binding to and activation of ClpP were much weaker thanthose of ADEP (1; Kapp = 12 nm, Hill coefficient: 2.2). However,the ADEP (1) and all fragments tested exhibited modest posi-tive cooperativity in ClpP binding (i.e. , Hill coefficients >1),suggesting that the ADEP and fragments bind in the samegeneral fashion.

To complement our finding that fragments activate ClpP invitro, we used chemical proteomics experiments utilizing frag-ment-derived, affinity reagents.[8] The N-acyldifluorophenylala-

Scheme 1. Fragments of a cyclic acyldepsipeptide have differential anti-bacterial activity against B. subtilis.

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nine propargyl ester (21) was coupled to a biotin-azide conju-gate via a CuI-catalyzed click reaction.[9] The reaction productwas bound to avidin–agarose beads to yield an affinity matrixthat was used to capture proteins from cell lysates of the wild-type and clpP-spx null strains of B. subtilis. The specificallybound proteins were eluted by using free fragment 5 andidentified by MASCOT proteomic analysis. We were gratified to

find ClpP among the 39 unique and functionally annotatedproteins that were positively identified (Table 1, Supporting In-formation). Interestingly, 20 of these proteins were also cap-tured from the lysates of the clpP-spx null strain. The biologicalsignificance of these off-target binding events is not clear, ascompound 14 and other fragments had no effect on thegrowth of the clpP-spx null strain (i.e. , the compounds’ bio-

Scheme 2. Structures and antibacterial activities of N-acyldifluorophenylalanine fragment analogues against B. subtilis.

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activity is dependent on ClpP). A speculative explanation forthe off-target binding of the eighteen unique proteins in thelysates of the wild-type strain is that some of them are ClpP

substrates (or degradation products thereof) that were trappedwithin the captured tetradecamer.[10]

In conclusion, a truly remarkable example of perturbation ofprotein–protein interactions by a small molecule underlies theantibacterial activities of the ADEPs. Their binding to ClpPinduces significant changes in the quaternary structure[2b, c] ofthe enzyme, which enhance off-target activity[11] and precludesinteraction with AAA+ partners.[2a] It has been proposed thatbinding and activation of ClpP are predicated on the mimicryof IGF and LGF motifs of the AAA+ partners by the ADEP sidechain. Here, we report that only the N-acyldifluorophenylala-nine moiety of the ADEPs is required for their bioactivities.These results are especially notable in light of reports that anIGF tripeptide alone does not activate ClpP or interfere withthe binding of ClpP to ClpX.[3a] We believe that the essentialityof the N-acyldifluorophenylalanine moiety for both ClpP activa-tion and antibacterial activity can be reconciled with our re-cently reported finding that restriction of the peptidolactonedynamics improves activity. Specifically, the strengthenedtrans-annular hydrogen bonding between the macrocycle andthe side chain could enhance cell permeability and lock theside chain in a conformation for optimized ClpP binding.

Our definition of the ADEPs’ simple pharmacophore has im-portant implications. Firstly, the ease of fragment synthesis, rel-ative to the ADEPs, makes ClpP activators more accessible tothe growing number of groups interested in their mechanismof killing bacteria. In addition, the reported fragments are gen-erally stable and can be stored at room temperature for weekswithout any detectable degradation with two exceptions (9 :light sensitive; 19 : acid sensitive). Secondly, our findings pro-vide a starting point for fragment-based design of non-peptideactivators of ClpP. In this ligand-design strategy, weakly activefragments are structurally elaborated into higher affinity li-gands.[12] Although fragment-based drug design is a relativelynew, it has become widely appreciated as a powerful tool indrug discovery.[12d–f] The fragment-based design strategy isa viable alternative to screening libraries of compounds in thesearch for non-peptide ClpP activators.[13] Such efforts are moti-vated by concerns that the ADEP peptidolactone backbonecould have pharmacological liabilities that are often associatedwith peptides,[14] despite the fact that structurally optimizedADEPs effectively cure bacterial infections in mice andrats.[1c, d, g] Conveniently, the N-acyldifluorophenylalanine moietyis highly amenable to structural elaboration, as it possessesa reactive carboxylate functionality that can be easily coupledto a wide array of scaffolds and rapidly diversified. Work to de-velop more potent antibacterial agents by using this strategyis underway in our laboratories.

Acknowledgements

J.K.S. is the recipient of a National Science Foundation (NSF)CAREER Award. D.C.W. gratefully acknowledges a dissertation fel-lowship from the Brown University Department of Chemistry. Thework was supported in part by National Institutes of Health (NIH)grant GM-101988 to R.T.S. Expert technical support on NMR and

Figure 1. ClpP binding of key N-acyldifluorophenylalanine fragment ana-logues. Hydrolysis of a fluorogenic decapeptide substrate (15 mm) by B. subti-lis ClpP (25 nm) was assayed in the presence of increasing concentrations ofADEP fragments, and activity was fit to a cooperative binding model (Fig-ure S2) to determine apparent binding constants (Kapp) and Hill coefficients.

Table 1. Affinity-captured proteins from B. subtilis lysates encoded byfunctionally annotated genes.

2-succinyl-6-hydroxy- DnaB* MinC*cyclohexa-2,4-diene-1-carboxylate synthase*3-ketoacyl-ACP DnaI RapJreductase*a-galactosidase fengycin synthetase* ribosomal

protein B*aminoglycoside flagellar switch protein* RNA polymerase6-adenylyltransferase s-54 factor*AnsB formamidopyrimidine-

DNA glycosidase*SHCHC synthase*

aspartokinase II a-subunit fructose-1-phosphate SinRkinase

ATP-dependent nuclease GerC3* spore coatprotein

bacillomycin synthetase A* GltC* SpoVScell division ATP-binding gluconate kinase* SrfAA surfactinprotein* synthetasechemotaxis protein gramicidin S sythetase* SubAClpP heptaprenyl sucrase

diphosphate synthase*deoxyribodipyrimidine l-aspartase superoxidephotolyase* dismutaseDNA polymerase I* lysine decarboxylase* TreP

[a] Thirty-nine functionally annotated proteins positively identified byMASCOT Proteomic Analysis as specifically bound to N-E-2-heptenoyldi-fluoro-phenylalanine affinity matrix. See the Supporting Information fora complete protein list. Proteins captured from both wild-type and theclpP null strain of B. subtilis are indicated by asterisks. ClpP is highlightedin bold text.

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mass spectrometry were provided by Russell Hopson, Tun-Li Shen,and James Clifton at Brown University. We thank Prof. AlanGrossman of MIT for providing the B. subtilis strains.

Keywords: antibacterials · ClpP · fragments · peptides ·proteolysis

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D. W. Carney, C. L. Compton,K. R. Schmitz, J. P. Stevens, R. T. Sauer,J. K. Sello*

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A Simple Fragment of CyclicAcyldepsipeptides Is Necessary andSufficient for ClpP Activation andAntibacterial Activity

Just one piece: Through a combinationof genetic and biochemical experi-ments, we established that the N-acyldi-fluorophenylalanine side chain of cyclicacyldepsipeptide antibacterial agents isnecessary and sufficient for biologicalactivity : fragments activate ClpP andretain antibacterial activity. This findingsets the stage for the design of novelClpP activators.

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