Consequences of Depsipeptide Substitution on the ClpP Activation

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Letter

Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides Yangxiong Li, Nathan P. Lavey, Jesse A. Coker, Jessica E. Knobbe, Dat C. Truong, Hongtao Yu, Yu-Shan Lin, Susan L. Nimmo, and Adam S Duerfeldt ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00320 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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ACS Medicinal Chemistry Letters

Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides Yangxiong Li,‡,§ Nathan P. Lavey,‡,§,† Jesse A. Coker,‡ ,§,† Jessica E. Knobbe,‡ ,§,† Dat C. Truong,‡ ,§,† Hongtao Yu,┴ Yu-Shan Lin,┴ Susan L. Nimmo,§ and Adam S. Duerfeldt*,‡,§ ‡

Institute for Natural Products Applications and Research Technologies and §Department of Chemistry & Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, USA ┴ Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA 02155, USA KEYWORDS Caseinolytic protease P, acyldepsipeptide, depsipeptide substitution, antibacterial. ABSTRACT: The acyldepsipeptide (ADEP) antibiotics operate through a clinically unexploited mechanism of action and thus have attracted attention from several antibacterial development groups. The ADEP scaffold is synthetically tractable and deep-seated modifications have produced extremely potent antibacterial leads against Gram-positive pathogens. Although newly identified ADEP analogs demonstrate remarkable antibacterial activity against bacterial isolates and in mouse models of bacterial infections, stability issues pertaining to the depsipeptide core remain. To date, no study has been reported on the natural ADEP scaffold that evaluates the sole importance of the macrocyclic linkage on target engagement, molecular conformation, and bioactivity. To address this gap in ADEP structure–activity relationships, we synthesized three ADEP analogs that only differ in the linkage motif (i.e., ester, amide, and N-methyl amide) and provide a side-by-side comparison of, conformational behavior and biological activity. We demonstrate that while replacement of the naturally occurring ester linkage with a secondary amide maintains in vitro biochemical activity, this simple substitution results in a significant drop in whole-cell activity. This study provides direct evidence that ester to amide linkage substitution is unlikely to provide a reasonable solution for ADEP instability.

The need for antimicrobials that operate through new mechanisms of action is urgent and indisputable, as drug resistance in pathogenic bacteria is beginning to outpace our derivatization of current therapeutic classes. As a key regulator of virulence in infectious bacteria, and given its roles in mediating bacterial protein turnover and homeostasis, caseinolytic protease P (ClpP) has emerged as a promising new antibacterial target.1-3 Indeed, ClpP represents a unique target, in that both inhibition and activation of this protease have therapeutic utility, with each strategy affecting different aspects of bacterial pathogenicity.2 This provides an opportunity to determine the therapeutic potential of two orthogonal strategies on a single target, a rare phenomenon in drug discovery. ClpP is a cylindrical serine protease composed of two stacked heptameric rings, which form a tetradecameric complex containing a large proteolytic core.4-7 The primary physiological role of the ClpP system in bacteria is to maintain protein quality control and degrade transient regulatory proteins. 2 Under normal homeostatic conditions, ClpP proteolytic activity is regulated through interactions with ATP-dependent AAA+ cochaperones (e.g., ClpX and ClpA), which are responsible for the denaturation, delivery, and translocation of tagged (i.e., SsrA or pArg) substrates into the proteolytic core of ClpP (Figure 1).810 In the absence of ATP or cochaperones, ClpP is inactive and unable to degrade substrates larger than 5–6 amino acids, protecting the organism from unregulated proteolysis. 11

Polypeptide

Polypeptide

Figure 1. Therapeutically relevant orthogonal approaches to ClpP modulation.

Although both ClpP inhibition and activation provide therapeutically relevant outcomes, “turning-on” bacterial enzymes/pathways to impart antibacterial activity deviates substantially from the status quo of antibiotic development. Small molecule and natural product activators of bacterial ClpP have been discovered,1, 12-14 but the natural product acyldepsipeptides (ADEPs, Figure 2) remain the most promising leads identified to date. ADEP chemo-activation of ClpP results in detrimental effects on microbial fitness and a reduction in virulence.1, 3 Structure–activity relationship studies of the ADEP scaffold have produced extremely potent analogs against Gram-positive

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pathogens;15-19 however, poor physicochemical properties, a limited spectrum of utility, and susceptibility to efflux have hindered the clinical development of this class.1, 17 Specifically, hydrolysis of the ADEP depsipeptide ester under basic or acidic conditions has been a major concern regarding this natural product family.17, 20 In fact, recent studies report almost complete degradation of various ADEPs in Mueller– Hinton broth within 24 h; a surprising claim given the benign nature of this broth.20 A common approach to improve the stability of ester linkages is to simply replace the ester with an amide or N-methyl amide. Ester to amide substitution has demonstrated utility to improve not only stability, but also to decrease off target cytotoxicity, and enhance permeability of cyclic lipodepsipeptides.21-22 While ester to amide substitution has been investigated sporadically on ADEP analogs,18, 23 no direct systematic comparison of compounds differing in only the macrocyclic linkage type (i.e., -O-, -NH-, -NMe-) has been reported. Furthermore, all linkage substitutions reported to date have been conducted on “pre-rigidified” ADEP analogs, in which unnatural amino acids have been introduced into the macrocyclic core to enhance structural rigidity.18, 23 Incorporation of unnatural rigidification in addition to macrocyclic linkage substitution, imparts a multi-variable effect, which may introduce conflicting conformational strains and make it difficult to delineate the specific effect of linkage substitution. As such, to determine the sole effect of the linker on the naturally occurring ADEP macrocyclic core, we have synthesized three ADEP analogs that differ only in the linkage type and have evaluated these analogs to provide insight regarding target engagement, molecular conformation, permeation, and antibacterial activity. Synthesis of ADEP Analogs. For the purpose of these studies, we selected 1 as the parent compound (Figure 2A), as this ADEP 1) is synthetically tractable;24 2) represents one of the most potent synthetic ADEP analogs that maintains the natural depsipeptide core;17 3) has been implemented in a number of studies as a positive control for the development of ClpP activators;15, 19 and 4) has been extensively characterized by the ClpP research community.15, 17, 19 Utilizing a combination of two different convergent strategies,16, 24 ADEP analogs 1–3 (Figure 2) were synthesized and the preparation of the new ADEP analogs, 2 and 3, is described.

Figure 2. A) ADEP analogs synthesized and evaluated in this study. B) Target fragments for the convergent synthesis of 1–3.

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The convergent synthetic approach relies on the strategic disassembly of the ADEP scaffold into three fragments (i.e., tripeptide, linkage, and F-Phe-heptenamide fragments), which can all be modified to generate new ADEP analogs (Figure 2). Specifically, we recognized the potential to leverage this synthetic approach to access 2 and 3 via modification of the linkage fragment. To synthesize the -NH- fragment 6, the protected t-butylcarbamate of D-proline (N-Boc-D-proline) 4 was coupled to 5 utilizing common peptide coupling conditions to provide the desired N-Boc-dipeptide in 84% yield (Scheme 1). Likewise, the -NMe- fragment 6’ was prepared in rapid fashion from the known dual-protected N-methylated unnatural amino acid 7. Protection of 7 as the phenacyl ester followed by Boc removal provided the secondary amine 8 as the trifluoroacetic acid salt. Coupling of 8 to N-Boc-D-proline provided the requisite -NMefragment 6’ in 83% yield over 3-steps (Scheme 1). Scheme 1. Synthesis of requisite linkage fragments.

a) HATU, DIPEA, DMF, 84% b) 2-bromoacetophenone, K2CO3, (CH3)2CO; c) CF3CO2H, CH2Cl2; d) 4, HATU, DIPEA, DMF, 83% over 3-steps. Pac = Phenacyl

With the required linkage fragments in hand, the full assembly of ADEP analogs 2 and 3 (Scheme 2) could be completed. Prior to fragment elaboration, linkage fragments 6 and 6’ were exposed to trifluoroacetic acid in methylene chloride to remove the Boc protecting group. The resulting crude trifluoroacetic acid salts 9 and 9’ were coupled to the tripeptide fragment 1016 to yield the requisite linear pentapeptides (11/11’). These pentapeptides were converted to 15 and 15’, respectively, in acceptable yields through a 4-step sequence including two protecting group removals, ester activation, and a base-catalyzed intramolecular cyclization. As anticipated, the yield of this sequence was significantly tied to the intramolecular cyclization reaction, which was drastically improved by decreasing the addition rate of 14/14’ to the pre-mixed aqueous sodium bicarbonate/methylene chloride reaction solution. Simply altering the addition rate, improved the yield of the 4-step sequence from ~20% to 61% (15) and 78% (15’). A similar observation has been noted with -O- linked depsipeptide derivatives.16 Removal of the Cbz protecting group under acidic conditions provides the hydrochloride salt 16/16’ in near quantitative yields. Compounds 16 and 16’ were then coupled to N-Boc protected 3,5-difluoro-L-phenylacetic acid to provide 17 and 17’, respectively.


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Scheme 2. Fragment assembly and synthesis of ADEP analogs 2 and 3.

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a) CF3CO2H, CH2Cl2; b) HATU, DIPEA, DMF, 73% (11), 93% (11'); c) Zn, 70% aq. AcOH; d) C6F5OH, EDC-HCl, CH2Cl2; e) 4M HCl in Dioxane; f) 1M aq. NaHCO3:CH2Cl2, 61% (15), 78% (15') over 4-steps; g) Pd/C, 1 atm H2, CH3OH, aq. HCl, 98% (16), 98% (16'); h) BocPhe (3,5-F2)-OH, HATU, DIPEA, DMF; i) CF3CO2H, CH2Cl2; j) (E)-hept-2-enoic acid, HATU, DIPEA, DMF, 76% (2), 51% (3) over 4steps; Pac = Phenacyl.

Subsequent Boc removal under standard conditions and exposure of 18/18’ to (E)-hept-2-enoic acid under peptide coupling conditions provided the desired ADEP analogs 2 (X = NH) and 3 (X = NMe). With analogs 1–3 in-hand, we conducted a number of parallel studies to compare macrocyclic conformation and biological activity in both biochemical and wholecell assays. Bioactivity Evaluation. Similar to the natural cochaperones, ADEP binding results in the reorganization of ClpP and a subsequent widening of the proteolytic chamber. 1, 4 Upon chemo-activation of ClpP, however, proteolysis is hyper-activated and unregulated, and thus detrimental to bacterial survival. To biochemically compare the ClpP activation potential of each analog, we evaluated the ability of 1–3 to induce degradation of a self-quenching decapeptide (AbzDFAPKMALVPYNO2).15 ClpP-induced cleavage of the decapeptide between the aminobenzoic acid fluorophore (Abz) and 2-nitrotyrosine quencher releases fluorescence, which can be quantified with a fluorimeter. Compounds 1–3 were evaluated over an 11-point dose range to determine an apparent binding constant (Kapp) for ClpP activation. As shown in Table 1 (Figure S1), the order of potency is 1 > 2 > 3, with a ~2-fold difference in potency between 1 and 2 and an additional ~50fold drop between compounds 2 and 3. This demonstrates a significant structure–activity relationship for the ADEP cyclic peptide linkage on ClpP activation potency. However, it also suggests that substitution may be allowable, as a respectable level of activity is maintained, especially when comparing the ester (1) with the secondary amide (2) linkage. All three analogs exhibit a Hill Slope coefficient > 1, suggesting modest positive cooperativity in ClpP binding, a phenomenon indicative of ClpP activators that bind competitively to the co-chaperone IGF loop binding pocket.5 As shown in Table 1 (Figure S2), the ester linked ADEP (1) exhibits an improved stabilization of ClpP in melt experiments relative to both the -NH- (2) and NMe- (3) compounds. This result is in congruence with the Kapp values determined in the decapeptide degradation assay.

All three compounds were evaluated in broth microdilution minimum inhibitory concentration (MIC) assays against Bacillus subtilis. As shown in Table 1, the ester exhibited the greatest whole-cell activity (37 nM) followed by the amide (6.25 µM) and then the N-methyl amide (>25 µM). Therefore, while the biochemical activity and thermal stabilization studies suggest -NH- macrocyclic substitution to be commensurate with the natural depsipeptide (~2-fold difference), the whole-cell activity demonstrates a significant difference in efficacy (>150fold difference), presumably due to a decrease in the efficiency of membrane permeation.25-26 Table 1. Activity comparison between ADEP analogs. Compound

Kapp (µM)

t1/2 (min)a

ΔTm (°C)

(µM : µg/mL)c 0.037 : 0.027

H/D Exchange

MIC

1 (-O-)

0.037 ± 0.005

38

29.1

2 (-NH-)

0.085 ± 0.01

< 4b

22.8

6.25 : 4.6

3 (-NMe-)

4.39 ± 0.60

< 4b

12.7

>25 : >18.6

aAmide

hydrogen of the alanine residue within the cyclic peptidolactone/peptidolactam core at 40 °C in CD3OD. bExchange was complete prior to the first scan. cBacillus subtillus (ATCC 6051).

Conformational Analysis. Inspection of the published ADEP1•ClpP (PDB ID: 3KTI) and ADEP2•ClpP (PDB ID: 3KTK) co-crystal structures reveals that the oxygen comprising the ester linkage of the depsipeptide is not involved in either intramolecular or intermolecular interactions when bound to ClpP.4, 17 As such, we hypothesized that any observed differences in biological activity, especially between the -O- and NH- linkages likely arises from conformational differences. To evaluate the effect of linkage substitution on the conformational dynamics of the cyclic peptidolactone/peptidolactam cores, we conducted hydrogen–deuterium exchange experiments. Specifically of interest, were the H/D-exchange properties of the alanine residue within the cyclic peptidolactone/peptidolactam cores. Intramolecular hydrogen bonding of the alanine -NH-



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and the extracyclic 3,5-difluorophenylalanine carbonyl is observed in ADEP•ClpP co-crystal structures and thus has been postulated as an important feature in ADEP binding.15, 17 Strengthening this intramolecular hydrogen bond interaction can be accomplished by incorporation of a pipecolate residue in the depsipeptide core.15 This structural modification leads to more rigidified analogs that mimic the bound conformation, and thus reduces entropic binding penalties, resulting in the observed improvement in potency for rigidified ADEPs.15 The -O- linked compound (1) exhibited a much longer H/D exchange rate (t1/2 = 38 min) than the -NH- (2) and -NMe- (3) compounds, both of which revealed complete H/D exchange within the time required to set-up the experiment (t1/2 < 4 min). This suggests that both the -NH- and -NMe- linkages perturb the macrocyclic conformation enough to disrupt the important intramolecular hydrogen bonding interaction between the alanine and the 3,5-difluorophenylalanine. This is noteworthy in regards to the whole-cell activity, as intramolecular hydrogen bonding of macrocycles has been shown to enhance permeation through lipid bilayers, presumably by decreasing energetic penalties for desolvation of hydrogen bond donors, especially NH groups.25-26 Thus, although the overall effect of macrocyclic conformation for -O- to -NH- substitution in the linker is relatively small and can be easily overcome in the biochemical assay, it is plausible that the disruption of this intramolecular hydrogen bond negatively affects permeation and contributes significantly to the decreased whole-cell activity of 2. Not surprisingly, the -NMe- linkage results in more drastic deviations from the optimal macrocyclic conformation, disrupting the intramolecular hydrogen bonding and likely producing a large amount of cis amide conformer, which cannot easily be overcome during binding, thus resulting in significant decreases in both potency and whole-cell activity. Indeed, our NMR analysis strongly indicates a conformational mixture of multiple highly populated conformations for 3 (see SI). To confirm that the conformational alteration resulting from the -NH- linkage (2) was likely limited to minor perturbations and not more significant events like amide bond or proline isomerization, we conducted a detailed analysis of amide bond geometries contained within the macrocyclic core and compared these results to those obtained for the -O- linked compound (1). Fortunately, for both 1 and 2, a single conformation was observed in both DMSO-d6 and CDCl3, facilitating the comparative analysis.

Figure 3. Numbering used for NMR discussion.

It has been long established that the conformation around the amide bond of a proline ring can be determined by the difference in the 13C and 13C chemical shifts of the proline ring.27-29 = 0–4.8 ppm is predicted to be 100% trans, where = 9.15– 14.4 ppm is predicted to be 100% cis. For ranges in between 4.8–9.15 ppm the data is ambiguous and should be confirmed by conventional NOE. A cis-peptide bond Xaa-Pro is indicated by a strong H–H NOE and a trans-peptide bond by a strong

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H–H NOE. Additionally, the splitting pattern of the H proton of the proline ring is indicative of cis or trans. The cis-proline H is a doublet or doublet of doublets with a much smaller second coupling constant and the trans-proline H is a multiplet or triplet.30 This difference in multiplicity is due to differences in vicinal coupling between H and the two Hprotons. 1H,13CHSQC-TOCSY, and 1H,13C-HMBC were used to unambiguously assign the carbon and proton chemical shifts of the proline rings. Both proline rings in 1 and 2 have cis conformation about their amide bonds. This is evidenced by all three of the above criteria. The H16 (Figure 3) proton is a doublet, the difference in chemical shift of C17 and C18 () is 9.4 ppm for 1 and 10.5 ppm for 2. Both 1 and 2 show a strong NOE between H16 and H21. H29 is a doublet of doublets, with coupling constants of 8.8 Hz and 2.2 Hz for 1 and 2. The difference in chemical shift of C30 and C31 () is 7.8 ppm for 1 and 7.5 ppm for 2 which is more ambiguous; however, there are strong NOE correlations between H29 and H12. The cis and trans conformation about the other amide groups were determined by NOE data. A cisamide will show a large NOE between H–HBoth 1 and 2 had the same following NOE correlations:The lack of NOE between H21 and H25 suggests a trans-conformation at H23; 2) The large NOE between H25 and H29 reflects a cis-conformation at the methylated nitrogen; 3) There is no NOE between H12 and H3, suggesting a trans-amide at H1. Additionally, for 2 no NOE was observed between H16 and H13, which suggests a trans-conformation at H14. Computational Analysis. In addition to H/D exchange experiments, we subjected the compounds to in silico conformational analysis. Molecular dynamics (MD) simulations utilizing an enhanced sampling method (bias-exchange metadynamics)31-32 were performed for 1 and 2 (in H2O). Details on the MD protocol and the conformational density profiles of the two compounds can be found in the supporting information. The major predicted conformation for 1 adopted a structure very similar to that seen in the co-crystal structure (PDB ID: 3KTI; backbone RMSD ~0.60 Å) (Figure 4A). On the other hand, 2 adopted multiple conformations in water, with the majority exposing the alanine -NH- to the solvent and thus lacking the intramolecular hydrogen bond between the alanine -NH- and the extracyclic 3,5-difluorophenylalanine carbonyl (Figure 4B). These results are consistent with the H/D exchange experiments, and biological activity. Conclusion. In summary, we have synthesized and biochemically evaluated three ADEP analogs that only differ in the type of linkage (i.e., -O-, -NH-, and -NMe-). This systematic study allowed for the direct comparison of linkage substitution on target engagement, conformation, and whole-cell activity. In biochemical activity assays, the -O- linked analog (1) exhibits ~2fold and ~100-fold better potency than the -NH- (2) and -NMe(3) analogs, respectively. In MIC experiments against Bacillus subtilis, 1 is ~170-fold and >650-fold more active than 2 and 3, respectively. In all biochemical assays in which these derivatives were evaluated, this 1>2>3 trend of activity was observed. Computational and spectroscopic analyses revealed that conformation is likely the key factor in differentiating target engagement and whole-cell activity. We demonstrate that simple replacement of the naturally occurring ester linkage with a secondary amide may not severely compromise the in vitro biochemical activity (target engagement), but results in a significant drop in whole-cell activity, presumably due to a disruption


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of a key hydrogen bonding interaction that is critical to cell permeation. As such, the excellent potency exhibited by rigidified ADEPs may not only arise from the pre-organization of the depsipeptide core into a conformation optimized for ClpP binding, but also from an enhanced permeation profile. This study provides direct evidence that ester to amide linkage substitution is unlikely to yield a solution to ADEP instability and further highlights the need to continue the exploration for new ClpP activating chemotypes.

Experimental procedures, characterization data, supplemental biological data, details on molecular dynamics simulations, and spectra (PDF)

AUTHOR INFORMATION Corresponding Author * Phone: (405) 325-2232. Fax: (405) 325-6111. Email: [email protected]

Author Contributions †N.P.

Lavey, J.A. Coker, J. Knobbe, D. Truong contributed equally to this work.

ACKNOWLEDGMENTS This work was partially funded by the Oklahoma Center for the Advancement of Science and Technology (OCAST, HR15-161) and University of Oklahoma start-up funds. Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103640).

ABBREVIATIONS AcOH, acetic acid; ADEP, acyldepsipeptide; ATP, adenosine triphosphate; Boc, tert-butyloxycarbonyl; Cbz, carbobenzyloxy; CDCl3, deuterochloroform; ClpP, caseinolytic protease P; DCC, dicyclohexylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDC-HCl, N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; HATU, O-(7-azabenzotriazol-1-yl)-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine; Pac, phenacyl; SAR, structure–activity relationship; TFA, trifluoroacetic acid; Zn, Zinc.

REFERENCES

Figure 4. Simulation results of A) compound 1 and B) compound 2. The cluster is shown in gray licorice and 100 structures selected from the cluster are depicted as thin blue thins (1) or red lines (2). Predicted intramolecular hydrogen bond between the alanine -NHand the 3,5-difluorophenylalanine carbonyl is indicated as a green dashed line. RMSDs are backbone deviations from PDB ID: 3KTI.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI 10.1021/acsmedchemlett.XXXXXXX.

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Graphical Abstract


Consequences of Depsipeptide Substitution on the ClpP Activation

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