among the most interesting 
 antibacterial drug targets to emerge 
 in the past decade is the proteolytic complex formed by clpp ( caseinolytic 
 peptidase ) and its aaa+ partners ( atpases associated with diverse cellular activities ) . clpp is a highly conserved peptidase that is involved 
 in the turnover of a wide variety of cellular proteins , including 
 transcription factors that regulate virulence - factor production and 
 stress responses . to form the catalytically active peptidase , clpp monomers self - assemble 
 into heptameric rings that stack face - to - face to form a barrel - shaped 
 tetradecamer . the  barrel  encloses a solvent - filled 
 chamber that is decorated with fourteen serine protease active sites 
 and , in principle , is large enough to accommodate a 50 kda protein . however , narrow axial pores at each end of the barrel prevent entry 
 of folded proteins into the proteolytic chamber . in fact , only 
 small peptides with 6 or fewer amino acids may freely diffuse into 
 the clpp proteolytic chamber and be degraded . in the degradation of 
 folded proteins , clpp functions in conjunction with aaa+ partners 
 like clpa , clpx , and clpc that recognize , unfold , and coaxially translocate 
 substrates into the proteolytic chamber . genetic studies 
 have established that the clpp gene and genes encoding 
 the aaa+ partners are essential for virulence in some pathogenic bacteria 
 ( e.g. , staphylococcus aureus , listeria monocytogenes , and streptococcus pneumoniae ) and for viability 
 in others ( e.g. , mycobacterium tuberculosis ) . although no drugs that target clpp have been introduced into the 
 clinic yet , the critical physiological roles of clpp make it an attractive 
 target for the development of antibacterial agents . several 
 molecules reported to perturb clpp activity have been discovered 
 in high - throughput screens or in mechanistic investigations of natural 
 products with antibacterial activity . treatment of bacteria with inhibitors of clpp 
 phenocopies the effects of clpp null mutations ( i.e. , 
 compromised virulence or viability ) , whereas bacteria 
 are killed upon exposure to clpp activators . the first clpp activators to be reported were the cyclic acyldepsipeptide 
 antibiotics ( adeps ) . produced 
 by streptomyces hawaiiensis(24 ) and enopeptins a and b produced by streptomyces sp . collectively , the adeps have been reported to exhibit potent 
 activity against a broad range of gram - positive bacterial pathogens , 
 including s. aureus , s. pneumoniae , enterococci , and m. tuberculosis . as reflected by the fact that no clinically used antibacterial drugs 
 target clpp , the adeps have activity against multidrug resistant , 
 pathogenic bacteria observed in clinical and community settings . structural studies indicate that 
 the adeps bind at the subunit 
 interfaces of the clpp tetradecamer , which also serve as docking sites 
 for the accessory atpases . a consequence of this 
 competitive binding is expansion of clpp s axial pores . remarkably , these adep - induced changes in clpp s 
 quaternary structure enable it to degrade oligopeptides and unstructured 
 or nascent proteins without the intervention of the accessory atpases . 
 the indiscriminate degradation of cellular proteins like the essential 
 cell - division protein , ftsz , by adep - activated clpp underlies the 
 antibiotics toxicity . with respect 
 to mechanism , the adeps are unique because most antibiotics inhibit 
 rather than activate their targets . the efficacy of adeps at 
 killing pathogenic bacteria and their 
 peculiar mode of action have prompted efforts to assess their medicinal 
 potential . initially , the natural products were found to be inactive 
 in mouse models of systemic s. aureus infection , 
 despite their potent antibacterial activity in vitro . their pharmacokinetic profiles were 
 characterized by poor water solubility , rapid systemic clearance , 
 and chemical instability . at bayer healthcare ag , a medicinal chemistry 
 program was established to optimize the structures of the adeps in 
 ways that would enhance their stability and biological activity . a compound called adep-4 , which had 160-fold 
 greater potency and more chemical stability than the natural products , 
 emerged from their optimization program . it differs from enopeptin 
 a and a54556a in three ways ( figure 1 ) . first , it has a more chemically stable heptenoyl 
 moiety in place of the conjugated polyenes . second , rather than phenylalanine 
 in its appendant side chain , adep-4 has a 3,5-difluorophenylalanine , 
 which was credited with improving compound bioavailability and binding 
 to clpp . finally , the natural adeps have a n - methylalanine 
 residue within the core macrocycle , whereas adep-4 has a cyclic amino 
 acid , pipecolate ( a six - membered ring ) , at the same position . accordingly , 
 it was proposed that the incorporation of the pipecolate residue rigidifies 
 the adep peptidolactone , thereby reducing the entropic cost of clpp 
 binding . this rigidifying structural 
 feature must enforce a specific conformation that is compatible with 
 clpp binding , as evidenced by the inactivity of an adep analog with 
 a proline residue ( a five - membered ring ) in place of n - methylalanine . in addition to potent 
 activity in vitro against s. aureus , s. pneumoniae , and enterococci , adep-4 and close analogs are reported 
 to be toxic to m. tuberculosisin vitro , particularly in combination with efflux pump inhibitors . in fact , mice with potentially lethal infections 
 of s. aureus were cured by via intravenous administration 
 of adep-4 . recently , it has been reported 
 to completely eradicate s. aureus biofilms in vitro and in mouse models of chronic infection when coadministered 
 with the antibacterial drug rifampicin . in a previous study , we investigated 
 the consequences of replacing amino acids in the adep macrocycle with 
 more conformationally constrained residues . one of the compounds that 
 we prepared was an analog of adep-4 with 4-methylpipecolate in place 
 of the pipecolate . the methyl substituent was predicted to further 
 restrict the conformational flexibility of the pipecolate and by extension 
 that of the peptidolactone . the compound with 4-methyl pipecolate 
 was 2- and 4-fold more potent than adep-4 against clinical isolates 
 of methicillin - resistant s. aureus and vancomycin - resistant enterococcus faecalis , respectively . motivated by these initial findings , we sought to study 
 the phenomenon of restricting peptidolactone conformation and define 
 its impact on the adeps binding to and activation of clpp 
 and on their bioactivity . we utilized deuterium exchange experiments 
 with h nmr to empirically measure the effects of various 
 structural modifications on peptidolactone conformational dynamics . in addition , enzymatic assays were used to measure the capacity of 
 the adep analogs to bind and activate clpp . finally , we used bioassays 
 to assess the toxicities of the compounds to three species of pathogenic 
 bacteria . it is noteworthy that some of the rigidified adeps bind 
 and activate clpp at substantially lower concentrations in 
 vitro and have up to 1200-fold enhanced antibacterial activity . numerous noncovalent interactions between the adeps 
 and clpp are observed in crystal structures of the complex . the adeps themselves adopt a compact conformation that appears to 
 be enforced by two transannular hydrogen bonds between the peptidolactone 
 and the appendant side chain ( figure 2a , table s1 ) . interestingly , a similar conformation 
 is observed in crystals of free adep , where analogous hydrogen bonding 
 between the peptidolactone and the side chain has been predicted . the similarities suggest that the free adeps 
 may be predisposed to adopt a conformation that is compatible with 
 clpp binding . ( a ) stereocartoon 
 of 
 an adep ( gray ball - and - sticks ) bound to escherichia coli clpp ( adjacent subunits in green and orange ) , generated from crystal 
 structure 3mt6 . two predicted hydrogen bonds are observed 
 within the adep ( black ; distances in  ) , and several hydrogen 
 bond networks ( cyan ) occur either directly between the adep and clpp 
 or via ordered water molecules . amides 
 participating in bonds are highlighted in blue and the nonbonding 
 amide is highlighted in red . the half - lives of the hydrogens of the 
 alanine and difluorophenylalanine residues were 26.8 and 3.87 min , 
 respectively ( see supporting information ) . to test the prediction that free 
 adeps exhibit intramolecular hydrogen - bonding 
 in solution , we performed experiments in which h nmr was 
 used to measure deuterium exchange rates of amide hydrogen atoms predicted 
 to participate in the bonds ( figure 2b ) . deuterium 
 exchange rates have been shown to be dependent upon the presence and 
 strength of intramolecular hydrogen bonds in peptides . accordingly , we anticipated that deuterium exchange 
 rates at the amides engaged in hydrogen bonds would be markedly slower 
 than at non - hydrogen - bonded amides . given the limited solubility of 
 the adeps in water , we selected deutero - methanol ( cd3od ) 
 as the solvent for the deuterium exchange experiments , accepting the 
 possibility that the molecules conformations could differ 
 in organic and aqueous solvents . immediately after preparation of 
 a dilute solution of adep 1a in cd3od ( figures 2b and 3 ) , we monitored attenuation 
 of the amide proton resonances by h nmr over a period 
 of hours at 25 c . as expected , the hydrogen atoms of the three 
 secondary amides in the adep exchanged with deuterium at markedly 
 different rates ( figure 2b , c ) . the amide hydrogen 
 of the serine residue , which does not participate in a transannular 
 hydrogen bond , exchanged completely in cd3od within several 
 seconds and could never be observed in a h nmr spectrum . the n - methylalanine , 
 pipecolate , 4-methylpipecolate and 4-isopropylpipecolate residues 
 are highlighted in red . the amide hydrogen of the side chain difluorophenylalanine 
 residue required several minutes to completely exchange with deuterium ; 
 whereas , that of the alanine amide within the macrocycle exchanged 
 over the course of two hours ( figure 2c ) . these 
 observations are consistent with the existence of transannular hydrogen 
 bonds that are analogous to those inferred from the crystal structures 
 of both free adep and adep in complex with clpp . we hypothesized that the bioactive conformation 
 of the adeps could be stabilized by judicious replacement of particular 
 amino acid constituents of the peptidolactone with more conformationally 
 constrained analogs . first , we envisioned substituting the n - methylalanine residue with pipecolate residues bearing 
 c-4 substituents of varying size . cyclic amino acids are typically 
 more conformationally constrained than their acyclic counterparts , 
 and ring substituents tend to limit ring conformational dynamics by 
 imposing high energetic penalties to certain conformations ( e.g. , 
 1,3- diaxial strain ) . in a separate approach , we envisioned replacing 
 the serine residue of the macrocycle with allo - threonine , 
 a serine analog with a methyl substituent on the -carbon . this 
 amino acid is more conformationally constrained because the methyl 
 group confers additional torsional strain about both the c-c 
 bond and c-o bond . importantly , we predicted that the methyl 
 substituent of this diastereomer of threonine would not sterically 
 clash with clpp . there are multiple precedents for the chemical 
 syntheses of the cyclic acyldepsipeptide antibiotics and analogs thereof . the desired adep analogs were 
 synthesized via a convergent strategy that was previously developed 
 in our laboratories ( see supporting information ) . the key tripeptide fragments containing 
 pipecolate or the substituted pipecolates were prepared using joulli - ugi 
 multicomponent reactions of dehydropiperidines , a chiral isocyanoacetate 
 derived from alanine , and boc - proline . an adep with 
 a natural product peptidolactone and an additional six adeps with 
 conformationally restricted amino residues in the peptidolactone were 
 chemically synthesized ( figure 3 ) . with the desired adeps in hand we anticipated that the deuterium exchange rates for 
 the hydrogens of the amides engaged in the hydrogen bonds would be 
 dependent on the conformational freedom of the adep peptidolactone . for all seven adeps , the alanine amide hydrogens half - lives 
 in cd3od were calculated from the rates at which their 
 resonances in h nmr spectra attenuated relative to those 
 of a nonexchanging reference signal in the same spectra ( figure 4 ; table 1 ) . compound 1a , a known molecule that is 
 the closest analog of the natural product enopeptin b with n - methylalanine and serine residues in its peptidolactone , 
 was expected to have the least rigid macrocycle and thus served as 
 a point of comparison for the other adeps . deuterium 
 exchange rates were measured for 2 mm solutions of each adep under 
 pseudo - first order conditions in deuterated methanol at 25 c . the exchange rates for the hydrogen atoms of the alanine residues 
 within the peptidolactone are shown . the bacterial 
 concentrations ( colony 
 forming units / ml ) in each well of the dilution antimicrobial susceptibility 
 tests were as follows : s. aureus ( 1.20  10 ) , e. faecalis ( 8.5  10 ) , and s. pneumoniae ( 3.65  10 ) . the deuterium exchange rates for the hydrogen atoms of the alanine 
 residues in the macrocycles are shown . using deuterium - exchange experiments , we systematically 
 assessed 
 the conformational consequences of replacing the n - methylalanine and serine residues in the adep macrocycle with conformationally 
 constrained pipecolate and allo - threonine residues , 
 respectively ( figure 4 ) . interestingly , the 
 pipecolate moiety does not fortify both of the transannular bonds 
 that are apparent in compounds containing n - methylalanine 
 ( compounds 1a and 1e ) . indeed , we found 
 that the amide hydrogen of the difluorophenylalanine of compound 1b exchanged completely within seconds in cd3od , 
 whereas the analogous hydrogen in compound 1a had a half - life 
 of 3.87 min ( see supporting information ) . apparently , with the pipecolate residue in the macrocycle , the 
 potential donor and acceptor atoms of the hydrogen bond are either 
 too far apart or do not have appropriate trajectories for bonding . in contrast , the transannular hydrogen bond in which the alanine residue 
 is the donor is retained in all of the adeps and strengthened by the 
 presence of conformationally constrained amino acids within the macrocycle . for instance , compound 1b harboring a pipecolate residue 
 in the peptidolactone had a slower rate of deuterium exchange rate 
 than 1a . further , we found that the deuterium - exchange 
 rate decreased as the steric bulk of the c4 substituent on the pipecolate 
 increased ( see data for compounds 1b , 1c , and 1d in figure 4 ) . replacement 
 of the native serine residue in the adep peptidolactones with allo - threonine profoundly slowed the deuterium - exchange 
 rate . for instance , the half - lives of the alanine amide hydrogens 
 in compound 1a , which has serine , and compound 1e , which has allo - threonine , are 100-fold 
 different . likewise , the deuterium exchange rate of the hydrogen atom 
 of the difluorophenylalanine moieties in compounds 1a and 1e differed by 2.8-fold . as expected , inclusion 
 of both pipecolate and allo - threonine ( 1f ) into the peptidolactone had a synergistic effect on deuterium exchange . interestingly , the apparent relationship between rigidifying structural 
 features and deuterium exchange rate was not completely conserved 
 when 4-methylpipecolate and allo - threonine were present 
 together in the peptidolactone ( 1 g ) . this compound had 
 a faster rate of deuterium exchange than compounds with allo - threonine and either n - methylalanine ( 1e ) or pipecolate ( 1f ) in the peptidolactone . a reasonable 
 explanation for this observation is that substituted pipecolate residues 
 and allo - threonine each stabilize slightly different 
 low energy conformers . accordingly , the opposing forces could prevent 
 a single , low energy conformer from being reached . as expected , the 
 rate of deuterium exchange increased for all compounds at an elevated 
 temperature ( i.e. , 40 c ) , whereas the trend for relative rates 
 of deuterium exchange remained the same ( see supporting 
 information ) , suggesting that the observed effects are the 
 result of entropic factors . overall , the general trend represented 
 by these data supports our hypothesis that the incorporation of conformationally 
 constrained residues in the peptidolactone has a rigidifying effect . binding of either the adeps or the 
 regulatory atpases to clpp stabilizes an open conformation of the 
 peptidase pore and stimulates degradation of oligopeptides . on the basis of predictions that 
 the entropic costs of clpp binding would be lower for the conformationally 
 constrained adep derivatives , we expected that adep 
 modifications that enhance macrocycle rigidity would improve clpp 
 binding and activation in a commensurate fashion . to test this hypothesis , 
 we assayed clpp catalyzed hydrolysis of an internally quenched fluorogenic 
 decapeptide in the absence and presence of the adep derivatives . cleavage 
 between an aminobenzoic acid fluorophore and 2-nitrotyrosine quencher 
 in this substrate relieves quenching , resulting in increased fluorescence 
 that serves as a readout of peptidase activity . the capacities of 
 each of the compounds to activate clpp were assessed across a range 
 of concentrations , and the resulting activities were fit to yield 
 apparent dissociation constants ( figure 5a , 
 table 1 ) . as expected , 
 we found a generally strong and positive correlation between the potency 
 of the compounds as activators of clpp and their rigidity as assessed 
 in the deuterium exchange experiments . kapp values range from 7.5 m for compound 1a , the 
 parental compound having the least rigid macrocycle , to 1.1 m 
 for compound 1 g , which possesses a significantly more 
 rigid macrocycle . interestingly , compound 1d was a weaker 
 activator of clpp than compounds 1b and 1c , despite having a more rigid macrocycle . the bulky c4-isopropyl 
 substituent may be poorly accommodated by the clpp binding pocket . 
 nevertheless , structural modifications that rigidify the adep peptidolactone 
 can improve clpp activation up to 7-fold in vitro . hydrolysis of a fluorogenic decapeptide substrate 
 ( 15 m ) by e. coli clpp ( 25 nm ) was assayed 
 in the presence of increasing concentrations of adep compounds , and 
 activity was fit to a noncooperative binding model ( solid lines ) . error bars represent standard deviation among three replicates or 
 standard error of the fit . tighter apparent affinities correlate with 
 increased adep rigidity , with the exception of compound 1d . ( b ) adeps with greater 
 macrocycle rigidity compete more strongly with clpx for binding to 
 clpp . fold change in atpase activity of e. coli clpx ( 10 nm ) in the presence of e. coli clpp ( 50 nm ) was assayed over increasing concentrations of adeps , 
 compared to the activity of clpx alone , and was 
 fit as above ( no fit was obtained for 1d ) . more rigid 
 adeps better compete for binding to clpp and , thus , more effectively 
 relieve clpp - mediated repression of clpx atpase 
 activity ( table 1 ) . in addition to modulating the quaternary structure of the 
 clpp 
 tetradecamer , adeps , and the accessory atpases share the same binding 
 sites and are known to compete for binding to clpp . as the rigidified 
 adeps bound to clpp more tightly , we predicted that these compounds 
 would be stronger competitors for atpase binding . we assayed binding 
 competition by exploiting the observation that e. coli clpx atpase activity is depressed upon binding e. coli clpp . accordingly , we inferred competition from 
 the degree to which the adeps relieved depression of atp hydrolysis 
 by clpx ( figure 5b , table 1 ) . as expected , the 
 increased competition with clpx also indicates that the more rigid 
 adeps bind more strongly to their original binding site on clpp , rather 
 than to novel sites . interestingly , compound 1d did not 
 effectively compete with clpx , despite the observation that it activated 
 clpp peptidase activity to the same extent as compound 1a . again , this weak suppression of clpx atpase activity can most likely 
 be ascribed to poor accommodation of the isopropyl group in the clpp 
 binding pocket . the antibacterial activity 
 of each compound was assessed against three gram - positive bacterial 
 pathogens : s. aureus , s. pneumoniae , and e. faecalis . minimum inhibitory concentrations 
 ( mics ) were determined by broth microdilution assays ( table 1 ) . there were largely positive correlations between antibacterial 
 activity , peptidolactone rigidity , and the apparent clpp affinity . compound 1d , despite possessing 
 a significantly more rigid peptidolactone , exhibited clpp affinity 
 and antibacterial activity similar to that of compound 1a . again , the presence of the large isopropyl substituent on the pipecolate 
 residue most likely has a negative effect on binding to clpp and thus 
 antibacterial activity . in contrast , compound 1 g , bearing 
 both a 4-methylpipecolate residue as well as an allo - threonine residue , was not the most rigid compound ( compound 1f in table 1 ) , yet it exhibited the 
 most potent antibacterial activity in the series . while these exceptions 
 can not be completely explained , we do note that adeps whose amide 
 hydrogens of the alanine residues have half - lives of more than 20 
 h in the deuterium exchange experiments have the highest clpp affinities 
 and most antibacterial activity against all three species of bacterial 
 pathogens . to the best of our knowledge , compound 1 g has 
 the lowest mics of any adep reported to date . the antibacterial activity of 1 g was 32-fold more potent 
 against s. aureus , 600-fold more potent against e. faecalis , and 1200-fold more potent against s. 
 pneumoniae than compound 1a , which has the more 
 flexible peptidolactone of the adep natural products . the cyclic acyldepsipeptide antibiotics are a promising class of 
 antibacterial agents that act by binding and dysregulating the activity 
 of the clpp peptidase . reports by our group and others state that 
 the activities of these compounds can be dramatically improved by 
 replacing certain amino acid constituents of the peptidolactone core 
 structure with more conformationally constrained counterparts . it has been proposed that improvements in bioactivity are a consequence 
 of these amino acids capacity to stabilize a bioactive conformation 
 of the adeps , which incurs a lower entropic cost upon binding to clpp . herein , we present data indicating that replacement 
 of selected constituent amino acids in the adep peptidolactone core 
 does indeed stabilize a bioactive conformation . specifically , analyses 
 of adeps harboring conformationally constrained amino acids via deuterium 
 exchange experiments revealed that they exhibit the same hydrogen 
 bonds in solution that are inferred from the crystal structures of 
 an adep in complex with clpp . our finding that replacement of the n - methylalanine moiety of the adep natural products with 
 a pipecolate attenuates deuterium exchange of only one of the two 
 donors ( i.e. , amide hydrogen of alanine ) in the hydrogen bonds , whereas 
 the substitution of the natural serine with allo - threonine 
 suppresses deuterium exchange rates of both hydrogen bond donors indicates 
 the position of the conformationally constrained amino acid within 
 the macrocycle has important effects on molecular conformation . in 
 molecules with either one or two hydrogen bonds , the inverse correlations 
 between the number of constrained amino acids constituting the peptidolactone 
 and the rates of deuterium exchange indicated that the amino acid 
 substitutions lock the adeps into a conformation that is compatible 
 with clpp binding . the apparent enhancements of the conformationally 
 constrained adeps capacities to both activate clpp and compete 
 with its binding to the accessory atpase clpx corroborate the proposal 
 that a bioactive conformation has been fortified . these improvements 
 are also consistent with the proposal that there is a lower entropic 
 cost in the binding of the rigidified adeps to clpp . the latter point 
 is of particular interest because the commonly held view that rigid 
 ligands suffer a lower entropic cost in receptor binding than flexible 
 ones has recently been challenged by 
 cases wherein there are entropic penalties for ligand preorganization 
 in receptor  ligand interactions . in any case , the finding that clpp activation by the adeps was enhanced 
 by up to 7-fold via the introduction of conformational constraints , 
 while these same changes enhanced antibacterial activity by up to 
 1200-fold indicates that there are other factors involved . a likely 
 explanation is that the constrained compounds are more cell - permeable . indeed , peptides with enforced transannular hydrogen bonds exhibit 
 dramatically enhanced cell - permeability and oral bioavailability because 
 the bonding reduces the energetic costs of desolvation that accompanies 
 membrane penetration in aqueous environments . apparently , the conformational constraints that we have introduced 
 enhance the adeps intrinsic transannular hydrogen bonding 
 interactions that predispose them for both clpp binding and membrane 
 penetration . it is well - known that the conformational constraints 
 of macrocylic 
 molecules can be further enhanced by judicious introduction of substituents 
 on the ring . in this case , it is notable 
 that installation of small methyl substituents profoundly enhances 
 the affinity of a large macrocycle for its biomolecular receptor and 
 the molecules bioactivities . although replacement of hydrogen 
 atoms with methyl groups is common in structure  activity relationship 
 ( sar ) studies and medicinal chemistry optimization programs , the inclusion of a methyl group on a ligand 
 typically is deleterious or minimally improves receptor binding . indeed , 
 a recent analysis of published sar studies by jorgensen and co - workers 
 states that in 8% of cases the inclusion of a methyl group enhances 
 bioactivity 10-fold or better . in only 
 0.4% of cases did molecules with an additional methyl group have 100-fold 
 enhanced bioactivity . their analysis 
 also revealed that significant improvements in bioactivity are usually 
 the result of the methyl group s capacity to fill a hydrophobic 
 environment in the receptor and to influence the conformation of the 
 ligand . interestingly , the substituent effect strategy exploited in 
 medicinal chemistry is mirrored in the adep natural products themselves . specifically , enopeptin a , which has a 4-methylproline residue in 
 its macrocycle , has a 2-fold lower mic against s. aureus , s. pneumoniae , e. faecalis , and e. faecium than enopeptin b which has an unsubstituted proline 
 residue at the same position . in this 
 study , we found that the position of the methyl substituent on the 
 adep peptidolactone is very important . when comparing the adeps lacking 
 methyl substituents ( compounds 1a and 1b ) to analogs harboring either 4-methyl pipecolate ( compound 1c ) or allo - threonine ( compound 1e ) , we find that the allo - threonine residue exerts 
 the strongest influence over conformational dynamics , clpp affinity , 
 and bioactivity . furthermore , it should be noted that inclusion of allo - threonine in the adep peptidolactone improves the mic 
 10-fold , while inclusion of 4-methylproline improves the antibacterial 
 activity only 2-fold ( as evidenced in the reported mics of enopeptin 
 a and enopeptin b ) . in this case , 
 the unique characteristics of both the small molecule 
 ligands and their receptor facilitated in - depth studies of a receptor  ligand 
 interaction . observations and modulations of the conformational dynamics 
 of adeps were accompanied by measurements of their affinity for clpp 
 and antibacterial activity . a distinguishing feature of our multifaceted 
 study was the use of deuterium - exchange h nmr experiments 
 to assess relative differences in conformational rigidities of the 
 adeps . we have shown that in such cases , hydrogen bonding can be 
 exploited to study the effects of structural modification on conformational 
 rigidity . we anticipate that this approach to small molecule dynamics 
 could be applied to studies of many ligand  receptor interactions 
 because many small molecules that interact with biological macromolecules 
 exhibit transannular hydrogen bonds ( especially peptides ) . it is a 
 much simpler alternative to sophisticated multidimensional nmr experiments 
 wherein n- and c - labeled compounds are used 
 to assess the dynamics of small molecules . the adep analogs 
 reported herein constituted by the conformationally 
 constrained amino acids allo - threonine and 4-methylpipecolate 
 have some of the lowest mics ever reported for antibacterial agents . the most potent adep reported prior to this work , adep-4 , was reported 
 to cure s. aureus infections in mice and s. pneumoniae infections in rats with even greater efficacy 
 than linezolid , a clinically used drug . given that our optimized analogs have mics against s. pneumoniae and e. faecalis that are 200-fold lower than those 
 reported for adep-4 , it is tempting to speculate that a dramatically 
 lower and potentially safer dose of our most potent compound could 
 be efficacious in the treatment of infections caused by streptococci , enterococci , and potentially other gram - positive 
 pathogens . an added advantage of the optimized compounds reported 
 here with respect to drug development is that the key allo - threonine residue is much less expensive and easier to prepare than 
 the 4-methylproline constituent of adep-4 . the promise of these molecules is further enhanced by the observations 
 that peptides with strong transannular hydrogen bonds have enhanced 
 oral bioavailability . testing of these compounds in 
 animal models of infection is currently underway in our laboratories . 
 in total , our findings provide a compelling illustration of how the 
 pharmacological properties of natural products can be improved by 
 rational design . nmr samples were prepared by 
 dissolving thoroughly dried adep in ampule sealed cd3od 
 at a concentration of 2 mm . the adep in cd3od was promptly 
 transferred to a clean nmr tube , purged with an argon atmosphere , 
 then capped and sealed with parafilm before being placed into the 
 nmr spectrometer . the integration of the exchanging 
 amide signal of interest was calibrated to a nonexchanging reference 
 peak . each data set was normalized such that the integral of the amide 
 signal of interest in the first spectrum acquired was equal to 1.00 
 and designated as t0 . data sets were plotted 
 in microsoft excel as normalized integrals vs time . plotted data sets e. coli clpp bearing a c - terminal his6 tag and single - chain pseudohexameric e. coli clpx ( amino acids 62424 ) 
 were expressed and purified by metal affinity , anion exchange , and 
 gel - filtration chromatography as described . in vitro assays were performed at 30 c in pd buffer ( 25 mm hepes , ph 
 7.5 , 100 mm kcl , 5 mm mgcl2 , 1 mm dtt , 10% ( w / v ) glycerol , 
 10% ( v / v ) dmso ) using a spectramax m5 microplate reader ( molecular 
 devices ) . peptidase activation was measured by incubating 25 nm of 
 clpp tetradecamer and each adep analog with 15 m of an internally 
 quenched fluorogenic peptide substrate , abz - kaspvslgyd , incorporating a 2-aminobenzoic acid 
 ( abz ) fluorophore and 3-nitrotyrosine ( y ) quencher . peptide hydrolysis by clpp was monitored by following the increase 
 in 420 nm fluorescence upon 320 nm excitation . initial analysis of 
 peptidase data showed negligible cooperativity ; thus , data were fit 
 to a quadratic form of a noncooperative binding equation , assuming 
 14 equivalent adep binding sites per clpp tetradecamer . to assay 
 adep competition for clpx binding to clpp , 50 nm of clpp tetradecamer , 
 10 nm of clpx pseudohexamer , 0100 m 
 of adep and 2.5 of mm atp were incubated with an nadh - coupled atp 
 regeneration system . atp hydrolysis was 
 monitored by following the coupled disappearance of nadh , via decrease 
 in 340 nm absorbance . pseudohexameric clpx is functionally 
 identical to monomerically encoded clpx , and was used to ensure hexamer stability at low clpx concentrations . atpase data were fit as above , assuming two clpx binding sties per 
 clpp tetradecamer . mic determinations 
 were performed 
 in bsl2 + conditions at the new england center for research excellence 
 ( nerce ) in biodefense at harvard medical school following standard 
 dilution antimicrobial susceptibility testing protocols . following incubation with the bacteria and the 
 compound , each well the mic is determined to be the first set of replicate wells 
 of the dilution series exhibiting no growth when compared to the growth 
 control wells .
the cyclic acyldepsipeptide ( adep ) 
 antibiotics are a new class 
 of antibacterial agents that kill bacteria via a mechanism that is 
 distinct from all clinically used drugs . 
 these molecules bind and 
 dysregulate the activity of the clpp peptidase . 
 the potential of these 
 antibiotics as antibacterial drugs has been enhanced by the elimination 
 of pharmacological liabilities through medicinal chemistry efforts . 
 here 
 , we demonstrate that the adep conformation observed in the adep 
 clpp 
 crystal structure is fortified by transannular hydrogen bonding and 
 can be further stabilized by judicious replacement of constituent 
 amino acids within the peptidolactone core structure with more conformationally 
 constrained counterparts . 
 evidence supporting constraint of the molecule 
 into the bioactive conformer was obtained by measurements of deuterium - exchange 
 kinetics of hydrogens that were proposed to be engaged in transannular 
 hydrogen bonds . 
 we show that the rigidified adep analogs bind and 
 activate clpp at lower concentrations in vitro . 
 remarkably , 
 these compounds have up to 1200-fold enhanced antibacterial activity 
 when compared to those with the peptidolactone core structure common 
 to two adep natural products . 
 this study compellingly demonstrates 
 how rational modulation of conformational dynamics may be used to 
 improve the bioactivities of natural products .