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Jul 26, 2016 - (17) Pantothenate kinase (PanK), the first enzyme in the CoA biosynthesis pathway, which catalyzes the ATP-dependent phosphorylation of...
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Discovery of potent pantothenamide inhibitors of Staphylococcus aureus pantothenate kinase through a minimal SAR study: inhibition is due to trapping of the product Scott J. Hughes, Leanne Barnard, Katayoun Mottaghi, Wolfram Tempel, Tetyana Antoshchenko, Bum Soo Hong, Abdellah Allali-Hassani, David Smil, Masoud Vedadi, Erick Strauss, and Hee-Won Park ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00090 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on August 1, 2016

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Discovery of potent pantothenamide inhibitors of Staphylococcus aureus pantothenate kinase through a minimal SAR study: inhibition is due to trapping of the product Scott J. Hughes†,○, Leanne Barnard∆,○, Katayoun Mottaghi§, Wolfram Tempel§, Tetyana Antoshchenko‡, Bum Soo Hong§, Abdellah Allali-Hassani§, David Smil§, Masoud Vedadi§, Erick Strauss*,∆ & Hee-Won Park*,‡ †

Department of Pharmacology, §Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada ‡ Department of Biochemistry and Molecular Biology, Tulane School of Medicine, New Orleans, LA 70112, USA ∆ Department of Biochemistry, Stellenbosch University, Stellenbosch, 7600, South Africa

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ABSTRACT The potent antistaphylococcal activity of N-substituted pantothenamides (PanAms) has been shown to at least partially be due to the inhibition of Staphylococcus aureus’s atypical type II pantothenate kinase (SaPanKII), the first enzyme of coenzyme A biosynthesis. This mechanism of action follows from SaPanKII having a binding mode for PanAms that is distinct from other PanKs. To dissect the molecular interactions responsible for PanAm inhibitory activity, we conducted a mini SAR study in tandem with the co-crystallization of SaPanKII with two classic PanAms (N5-Pan and N7-Pan), culminating in the synthesis and characterization of two new PanAms, N-Pip-PanAm and MeO-N5-PanAm. The co-crystal structures showed that all the PanAms are phosphorylated by SaPanKII, but remain bound at the active site; this occurs primarily through interactions with Tyr240' and Thr172'. Kinetic analysis showed a strong correlation between kcat (slow PanAm turnover) and IC50 (inhibition of pantothenate phosphorylation) values, suggesting that SaPanKII inhibition occurs via a delay in product release. In-depth analysis of the PanAm-bound structures showed that the capacity for accepting a hydrogen bond from the amide of Thr172' was a stronger determinant for PanAm potency than the capacity to π-stack with Tyr240'. The two new PanAms, N-Pip-PanAm and MeO-N5-PanAm, effectively combine both hydrogen bonding and hydrophobic interactions, resulting in the most potent SaPanKII inhibition described to date. Taken together, our results are consistent with an inhibition mechanism wherein PanAms act as SaPanKII substrates that remain bound upon phosphorylation. The phospho-PanAm–SaPanKII interactions described herein may help future antistaphylococcal drug development.

Keywords: Pantothenate kinase, coenzyme A, pantothenamide, growth inhibition, X-ray crystallography, enzyme inhibition.

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INTRODUCTION Multidrug-resistant (MDR) bacterial infections have emerged as one of the top global health issues according to the World Health Organization (WHO), with at least 23,000 deaths per year in the United States resulting from drug resistant bacteria.1 Unfortunately, this rise in MDR negatively correlates with the number of FDA approved antibiotics.2,3 Methicillin-resistant Staphylococcus aureus (MRSA) infections, which account for nearly half the MDR-related deaths a year in the United States and are the most common healthcare associated infection,1 typically fall to a handful of treatment options including vancomycin, daptomycin and linezolid.4,5 As resistance has already emerged to these drugs5,6 and some require close patient monitoring due to their associated toxicities,7,8 the need for potent, species-selective antimicrobials that target novel pathways in S. aureus is acute.9 Recent advances in rapid pathogen identification10 have increased the appeal of species-selective antimicrobials that limit cytotoxicity and damage to the normal microbial flora of humans. As the major acyl carrier in all forms of life, coenzyme A (CoA, 1) is central to pyruvate oxidation and fatty acid biosynthesis—both key to cellular growth and proliferation.11,12 Additionally, CoA has been suggested to play a further role in the maintenance of redox balance in S. aureus, due to the absence of glutathione in this organism.13,14 Consequently, the universal series of five enzymatic reactions that is responsible for the biosynthesis of CoA from pantothenic acid (vitamin B5, Pan, 2) have all been considered as potential targets for antibacterial drug development (Fig. 1a), and even more so in S. aureus.15,16 The discovery of inhibitors of the fourth enzyme in the pathway (phosphopantetheine adenylyltransferase, PPAT), which show in vivo efficacy in S. aureus infection models, has validated this point of view.17 Pantothenate kinase (PanK), the first enzyme in the CoA biosynthesis pathway which catalyzes the ATP-dependent phosphorylation of Pan to 4'phosphopantothenic acid (P-Pan, 3), has similarly received considerable attention in several inhibitor development studies targeting a wide range of pathogenic bacteria (Fig. 1a).16 However, none of these have so far yielded promising lead compounds with potential for development as antimicrobial agents. The lack of success is partly due to shortcomings in our understanding of CoA biology, which is compounded further by the diversity among PanK enzymes affecting the regulation of CoA biosynthesis and availability.12 In this study we focused on using structure-directed binding analyses and kinetic characterization to discover selective SaPanKII inhibitors that show increased potency and that can increase our understanding of this enzyme’s unusual substrate specificity.

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The diversity of PanK enzymes is embodied by their occurrence in three types that have different structures, regulation mechanisms and active site architectures.12 Among bacteria, two types dominate: type I PanKs, such as those from Escherichia coli (EcPanKI) and Klebsiella pneumoniae (KpPanKI), which are promiscuous, accepting a range of Pan analogs—including pantetheine (PantSH, 4), the precursor of the CoA salvage pathway (Fig. 1a)—as substrates with equal efficiency,18,19 and type III PanKs, such as the Pseudomonas aeruginosa (PaPanKIII) and Bacillus anthracis (BaPanKIII) enzymes, which are highly selective for Pan.20-23 However, S. aureus stands out among bacteria in that it possesses one of the known examples of a prokaryotic type II PanK, which more closely resembles those found in eukaryotes.12 Moreover, SaPanKII exhibits several additional unusual characteristics, such as resistance to feedback inhibition by CoA and its thioesters24 as well as showing differentiation in its activity toward Pan compared to its amide analogs, the N-substituted pantothenamides (PanAms).25 Since they were first synthesized in 1970,26 the PanAms have been the most studied of all potential CoA-directed antimicrobial inhibitors. These compounds inhibit a variety of organisms,15 including the malaria parasite,27 but show no cytotoxicity to human cell lines in culture.28,29 While initially showing great potential, the finding that the PanAms are susceptible to degradation by pantetheinase enzymes present in serum raised concerns about their potential for clinical application.30 However, this liability has recently been addressed by studies showing that the PanAms can be protected against degradation by either the co-administration of pantetheinase inhibitors,29 or by the introduction of minor structural modifications—neither of which has negative cytotoxicity implications.28,31 Consequently, the attention has shifted to understanding the PanAm’s mode of action to provide a basis on which their potency can be improved though rational design. Recently, it has become clear that the mode of action of a PanAm in a specific organism depends largely on that organism’s PanK type: bacteria with highly specific type III PanKs are resistant to PanAm inhibition, while those that have PanKI enzymes that allow the salvage pathway to operate also accept PanAms as substrates.25 Various crystal structures of type I PanKs suggest that this substrate promiscuity stems from a small hydrophobic pocket beyond the Pan carboxylate that can accommodate the amide substituents of PanAms, thereby allowing them to bind for the phosphorylation reaction.18,19,32 This metabolic activation of the PanAms eventually results in the formation of CoA antimetabolites, which can inhibit a range of CoA-dependent processes. In E. coli the most likely basis for PanAm antibacterial action is the inhibition of fatty acid biosynthesis

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through the formation of inactive acyl carrier proteins (ACPs) from such CoA antimetabolites; instead of being modified with phosphopantetheine groups to form holo-ACPs, these proteins have phosphopantothenamide arms that lack the thiol required for their acyl carrying function (and are therefore referred to as so-called crypto-ACPs).33,34 In S. aureus, characterization of the PanAms’ mode of action has been less clear-cut due to often contrasting inhibition analyses. An initial study found that PanAms act by inhibiting SaPanKII directly,35 while subsequent work pointed to a complex inhibition mechanism through the formation of CoA antimetabolites and inactive ACPs and analogous peptidyl carrier proteins (PCPs).24,36 Recently, PanK inhibition was again highlighted as an important contributor to the antistaphyloccal effects of the PanAms when comparative kinetic analyses of EcPanKI and SaPanKII showed that the PanAms have a dual interaction mode with the latter enzyme, acting both as high-affinity substrates with a slow turnover rate and as uncompetitive inhibitors.25 Based on these results, a kinetic model was proposed in which the PanAms bind either in SaPanKII’s active site, or in an as yet uncharacterized allosteric site. However, the first structure of SaPanKII bound to a PanAm that was released soon after—that of N-homopiperonylpantothenamide (N-HoPip-PanAm 8, previously N354Pan, Fig. 1b)—surprisingly showed the phosphorylated PanAm product trapped in the active site.32 This information suggests an alternative inhibition mechanism, in which the PanAms act as the substrates of SaPanKII, but the resulting phosphorylated products remain bound in the active site, effectively blocking the binding of the natural substrate (Pan). Taken together, the results obtained on SaPanKII to date indicate that the enzyme distinguishes between Pan and the PanAms in a very specific manner. The co-crystal structure of SaPanKII bound to phospho-N-HoPip-PanAm (PDB ID: 4NB4) reveals several binding interactions with its

N-substituent that could form the basis for its selectivity.32

Since an improved

understanding of these interactions could guide the development of new, non-pantothenamide inhibitors of SaPanKII, we set out in this study to perform a structure activity relationship (SAR) study using the N-HoPip-PanAm structure and those of the classic PanAms N-pentyl- (N5-Pan, 6) and N-heptylpantothenamide (N7-Pan, 7) (Fig. 1b) as a starting point. This culminated in the design, synthesis and characterization of two new PanAm structures, which show the most potent inhibition of SaPanKII described to date.

By correlating the structural results with the

antistaphylococcal activity data, these studies may direct the future development of new antistaphylococcal agents to target CoA biology.

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RESULTS AND DISCUSSION SaPanKII treated with ATP and either N5- or N7-Pan: structures of the product-bound enzyme. To provide structural data beyond that which was provided by the N-HoPip-PanAm structure,32 we solved the co-crystal structures of SaPanKII treated with either of the two classic PanAms N5-Pan (6) and N7-Pan (7) (the latter being the most potent antistaphylococcal PanAm identified to date) in the presence of ATP (i.e., in the same manner that the co-crystal structure of SaPanKII bound to phospho-N-HoPip-PanAm and ADP was obtained). N5-Pan-bound SaPanKII was crystallized as a non-crystallographic homodimer, whereas the N7-Pan-bound homodimer was generated by applying a 2-fold symmetry operation (Table 1). Each subunit of the homodimers is composed of two domains (Domain I and II) and the binding cap (residues 153–173), with a dimerization interface along helix α6 (Fig. 2a). We noticed that in both cases the structures were of the product complexes, i.e. similar to the phospho-N-HoPip-PanAm structure.32 The phosphorylated PanAm products are contained in a common binding site that comprises a mixture of hydrophobic and hydrophilic residues from both subunits of the SaPanKII homodimer (Fig. 2b), with the Pan moiety (C1–C8 inclusive, Fig. 1b) bound in a groove between β6-β8 of the 1st subunit (residues 92– 117) and the binding cap of the 2nd subunit. The other main interactions of the Pan moiety are similar to those found in the phospho-N-HoPip-PanAm-bound structure: the C4 and C8 amide carbonyls form hydrogen bonds with the side chain of Arg113, while the amide nitrogen N5 is within hydrogen bonding distance of the backbone carbonyl of binding cap residue Ala173' (where prime indicates this residue is from the 2nd subunit) (Fig. 2b). Additionally, one of the geminal dimethyl groups on C2 points toward the lipophilic side chains of Val156' and Ala173' of the binding cap. The putative catalytic residue Glu70 was found within hydrogen bonding distance of the oxygen bearing the phosphoryl group, located just 3.2 Å and 3.6 Å away in the respective N5-Pan and N7Pan structures, lending further support for the role of Glu70 in catalysis.20

Furthermore, the

2+

phosphoryl group forms several interactions with ADP, Mg and water, while remaining ~4 Å from Ile159' of the binding cap. Last among common interactions, the amide nitrogen N9 that connects the Pan moiety to the N-substituent is within hydrogen bonding distance of the backbone carbonyl of binding cap residue Thr172' (Fig. 2b). The N-substituent of each phospho-PanAm occupies a cavity formed by Leu171' and Thr172' of the binding cap, Glu202' and Thr206' of helix α6' and Tyr240' of helix α7' of the 2nd subunit, an area which will be referred to as the “N-substituent binding site”. Specifically, the alkyl chain NHughes et al.

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substituents of N5-Pan (6) and N7-Pan (7) are encircled by residues Leu171', Thr172', Glu202' and Tyr240' (Fig. 2b). Furthermore, weak electron density for some aliphatic carbon atoms distal to the amide link suggests some degree of mobility and poor fixation of the alkyl moieties inside the PanAm N-substituent-binding site, as demonstrated by the observed disorder for N7-Pan’s substituent.

By comparison, in the phospho-N-HoPip-PanAm structure (PDB ID: 4NB432) the

phenyl moiety of the PanAm substituent forms a π–π interaction with Tyr240', while one of the ring oxygen atoms is involved in a hydrogen bond with the backbone amide of Thr172' (Fig. 2c). By further comparison, in the structure that only has the nucleotide analog AMPPNP bound (PDB ID: 2EWS20) the side chain of Tyr240' fills the cavity otherwise occupied by the PanAm N-substituents and its hydroxyl interacts with the backbone amide of Thr172' (Fig. 2d). Taken together, these structures implicate Tyr240' and Thr172' in playing crucial roles in the PanAm binding event through their movement and direct interaction with the PanAm substituents. We therefore set out to establish their respective contributions to binding (and inhibition) of SaPanKII through an SAR study. Design and synthesis of the PanAm series and their analogs for SAR study. The two main structural components of the 1,3-benzodioxole moiety of N-HoPip-PanAm (8) that determine the interactions with Tyr240' and Thr172' are the phenyl ring and the ether oxygen in the 3-position. We therefore designed a series of PanAms in which these components were either isolated—giving rise to N-[(3-methoxy)-phenethyl]pantothenamide (N-3-MeO-PE-PanAm, 9)—or step-wise removed, resulting in N-phenethylpantothenamide (N-PE-PanAm (10), which only retains the phenyl ring) and N-(5-methoxypentyl)pantothenamide (MeO-N5-PanAm (11), which only has the ether group) (Fig. 1b). Finally, the analog of 8 that retains the benzodioxole moiety but attached to a methylene instead of an ethylene group was prepared; N-piperonylpantothenamide (N-Pip-PanAm, 12) should be less flexible than 8, and should therefore be more likely to maintain the conformation required for binding in the N-substituent binding site while retaining all the other components necessary for interacting with Tyr240' and Thr172'. The new PanAms were synthesized by established methods (Scheme S1) and thoroughly characterized prior to use in the following biological assays (see Methods for details). SAR of SaPanKII inhibition by the PanAm series. To perform the SAR analysis of the PanAm series, their inhibition of SaPanKII activity was assessed by determining the PanAm concentration that leads to 50% inhibition of SaPanKII-mediated phosphorylation of 25 µM Pan (a concentration similar to the previously determined K0.5 value for Pan25), i.e. by obtaining IC50 values. The results Hughes et al.

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(Fig. 3a and Table S1) clearly show that the interaction with the backbone amide of Thr172' is significantly more important than the π–π interaction with Tyr240', as MeO-N5-PanAm (11), which retained only the hydrogen bond acceptor, is the most potent inhibitor of SaPanKII activity identified to date, while N-PE-PanAm (10), which can only form the π–π interaction, is the least potent member of the series. However, between these extremes the analysis is less clear cut, as the PanAms with more flexible alkyl substituents (like N5- and N7-Pan) are more potent inhibitors than 10, although none of these PanAms’ substituents have hydrogen bonding capability. Nonetheless, 11 and 12 have potency better than the other members, suggesting that factors other than binding interactions could be at play in determining their potency. In our previous kinetic study of bacterial PanKs interacting with PanAms we found that those compounds that show potent growth inhibition of S. aureus, also exhibit unusual SaPanKII activity profiles.25

Specifically, SaPanKII had

exceedingly low Km values (indicating high affinity), as wells as low kcat values (correlating with slow turnover and/or product release) for such PanAms. To investigate this further, we determined the kinetic parameters of the PanAm series with SaPanKII. PanAm inhibitory potency correlates with their slow turnover. We obtained the activity profiles of the PanAms 7–12 with SaPanKII using the PK/LDH coupled assay that measures NADH consumption following the production of ADP (Fig. 3b). From these profiles, the kinetic parameters were determined by regression analysis as was done previously25 (Table S1). To test if a correlation exists between the poor turnover of a PanAm and its inhibition of SaPanKII, the determined kcat values were plotted against the IC50 values (the data for 6 was from the previous study25). The resulting analysis (Fig. 3c) shows that with the exception of N5-Pan (6), an excellent linear correlation exists between the data sets, with the most potent inhibitors of SaPanKII activity (11 and 12) also showing the lowest kcat values. Taken together with the finding that treatment of SaPanKII with the PanAms 6, 7 and 8 and ATP yields co-crystal structures with their phosphorylated versions and ADP bound to the enzyme, the results suggest that these PanAms inhibit SaPanKII by acting as high affinity substrates, which are released from the enzyme at a slow rate once phosphorylated. We propose that the kcat values determined for the PanAms can be correlated with the off rate for their phosphorylated products, as the standard assumption in steady state enzyme kinetics that product release is much faster than the catalytic step does not hold for these compounds (see expanded discussion in Supporting Information).

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SaPanKII structures with MeO-N5-PanAm and N-Pip-PanAm are also of the phosphorylated product complexes. The correlation between the inhibitory potency and low turnover rate of the PanAms 11 and 12 suggested that they most likely inhibit SaPanKII by being trapped as their product complexes. To determine if this is indeed the case, the co-crystal structures with both of these PanAms were obtained in a manner similar to those of the other PanAm structures. Indeed, in both the structures we found the enzyme to be bound to the phosphorylated products and ADP, with the conformation and binding interactions of their phosphate and pantothenoyl moieties being nearly identical to those of PanAms 6–8 (Fig. 4a). As predicted, the ether oxygen of the N-substituent of MeO-N5-PanAm (11) forms a hydrogen bond with the backbone nitrogen of Thr172' of the binding cap; this reduces its mobility in the N-substituent binding site, and could be an important contributor to the increased binding efficiency of 11 compared to N5- and N7-Pan that are incapable of forming this hydrogen bond (Fig. 4b). Considering the large difference in inhibitory potency between N-PipPanAm (12) and N-HoPip-PanAm (8), we were surprised to find that their structures are nearly identical: the 1,3-benzodioxole group of either 8 or 12 forms a π-stack with Tyr240' and is within van der Waals distance to other encircling residues (Leu171’, Thr172’ and Glu202’), and the ring oxygen at position 1 forms a hydrogen bond with the backbone amide of Thr172' (Fig 4c). However, close inspection reveals that the N-Pip-PanAm substituent is more ideally positioned for π-stacking and van der Waals contacts than that of N-HoPip-PanAm, as the 1,3-benzodioxole ring of the former is nearly parallel to the plane of Tyr240' and positioned almost equidistant from Glu202' and Leu171' (3.7 Å and 4.1 Å, respectively).

By comparison, the extra methylene in 8 causes the 1,3-

benzodioxole ring to rotate ~35° from the plane of Tyr240', positioning it closer to Glu202' and the side chain carbon Cβ atom of Tyr240' (3.4 and 3.3 Å, respectively) than to Leu171' and the side chain carbon Cζ atom of Tyr240' (4.2 and 4.6 Å, respectively) (Fig 4d). Therefore, in 8, less favorable πstacking and VDW contacts with surrounding residues may offset the contribution of hydrogen bonding. Together, these structural and biochemical findings suggest that the high potency of PanAm inhibitors arises from the combination of optimal polar and nonpolar interactions with residues lining the N-substituent binding site; these interactions promote their high affinity binding as substrates and cause their subsequent slow release once phosphorylated. Comparison of the product-bound structures with SaPanKII bound to AMPPNP: determinants of substrate binding and product release. The low turnover rates observed for the PanAms compared to Pan suggest that important differences exist in the manner that SaPanKII binds to and

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interacts with these compounds as substrates, and in how their phosphorylated products are released. Currently, no co-crystal structure with Pan or P-Pan exists; however, in all the structures of SaPanKII obtained with the PanAms we observed that multiple residues from the binding cap interact with various parts of the resulting phospho-PanAm at the active site. This finding suggests that the binding cap could act as a gate for substrate entry and/or product release. To investigate the binding cap’s seemingly multifunctional roles, we compared the structure of SaPanKII bound to phospho-N-Pip-PanAm and ADP with that of the nucleotide analog AMPPNPbound structure (PDB ID: 2EWS20). This revealed that an apparent ordering of the binding cap occurs upon PanAm binding, which includes the formation of a one-turn helix within the binding cap (Fig. 5a and 5b). The ordered binding cap contains a small pocket of highly conserved residues that form nonpolar interactions with the geminal dimethyl groups of the Pan moiety of the incoming substrate (either Pan or the PanAms) (Fig. 5c). Specifically, the side chains of Val156' and Ala173' are within 4 Å of one of the geminal dimethyl groups. Given that Val156' is also the first residue in the one-turn helix, these nonpolar interactions formed upon PanAm binding may help to rigidify the binding cap, thereby closing the binding pocket to prevent substrate departure (Fig. 2b and 5d). In concert with helix formation, the highly conserved Tyr160' is brought within hydrogen bonding distance of the putative nucleophile Glu70 (Fig. 5d and 5e). This is likely to help position the carboxyl group of Glu70 near the primary hydroxyl group of the substrate for subsequent phosphorylation (Fig. 5e). The ordering of the binding cap coincides with Ile159' approaching to within 4 Å of the phosphoryl group of the phospho-N-Pip-PanAm product, an observation that may help understand the mechanism of product release (Fig. 5e). An opening near the adenine moiety of bound ADP opposite from the pantothenate moiety-binding site suggests that nucleotide entry and exit could occur independent of any movement in the binding cap, allowing for facile replacement of ADP with ATP. The γ-phosphate of the incoming ATP would repel the phosphoryl group of the phospho-Pan product, pushing it closer to Ile159'; this unfavorable interaction could lead to a dissociation of the binding cap from the substrate-binding pocket, allowing for product release.

However, in the

phospho-PanAm-bound structures this push for dissociation could be offset by the binding interactions between their N-substituents and Thr172'/Tyr240', which would disfavor the opening of the binding cap. The ability of the PanAms to interact with Thr172' and Tyr240' would therefore also provide an explanation for their low turnover compared to Pan. Such a conclusion is supported

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by the finding that in eukaryotic PanKs such as the human PanK3, which do not distinguish catalytically between Pan and the PanAm analog PantSH 4, Thr172' and Tyr240' are replaced by Val and Ala respectively (Fig. 5c).37 Correlation of PanAm inhibition of SaPanKII with their antistaphylococcal potency. To correlate PanAms’ inhibition potency on SaPanKII activity with their antistaphylococcal activity, we determined the minimal inhibitory concentrations (MICs) of PanAms against S. aureus in both minimal (i.e. devoid of Pan) and rich media. Gentamicin served as positive control. Our results are in agreement with those of previous studies, showing N7-Pan (7) to be the most potent inhibitor of S. aureus growth in all media tested with an MIC value as low as 20 nM in minimal medium (Table 2).33,35,36 However, the SaPanKII inhibitory potency of the newly discovered MeO-N5-PanAm (11) and N-Pip-PanAm (12) did not translate into whole cell activity, with both showing at least 50-fold increase in MIC compared to 7 in minimal medium and 1% tryptone. In fact, N-PE-PanAm (10), which was the least potent inhibitor of SaPanKII activity in the series (Fig. 3a and Table S1), showed more potent growth inhibition than either of these compounds. This suggests that the presence of the ether oxygen poses a significant impediment to cell penetration, leading to the reduced potency of the new PanAms. The fact that the PanAms all show improved inhibition in the minimal medium suggested that the high concentration of Pan in rich media counters their inhibitory effects. This was confirmed by showing that co-administration of 100 µM Pan with 12 in Mueller-Hinton broth results in a 2-fold increase in the MIC. This result is in agreement with what was found in a similar experiment with 6,38 and confirms that the PanAms inhibit Pan-dependent processes, of which PanK is the first potential intracellular target.

CONCLUSIONS In the present study, we performed a minimal SAR study and determined the co-crystal structures of SaPanKII with two classical PanAms (N5-Pan and N7-Pan) as well as two novel PanAms (MeO-N5-PanAm and N-Pip-PanAm) uncovered as the most potent inhibitors of SaPanKII activity described to date. Our results show that although the PanAms are phosphorylated by SaPanKII, a delay in product release—likely brought about by interactions of the pantothenamide Nsubstituent with residues in helix α7' (specifically Tyr240') and the binding cap (specifically Thr172') stabilizing the closed conformation of the latter—result in reduced turnover rates and consequently inhibition of SaPanKII activity toward the endogenous substrate Pan. This most likely forms the basis for the inhibition of SaPanKII activity by the PanAms observed to date. Hughes et al.

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The results presented here call into question a recently proposed kinetic model for SaPanKII indicating that the PanAms, apart from being substrates, are also uncompetitive inhibitors of SaPanKII binding in an allosteric binding site on the enzyme.25 This model was mainly based on kinetic results obtained with 4'-deoxy variants of PanAms that lack the -OH group that is phosphorylated by SaPanKII, and that therefore can only act as non-phosphorylatable substrate analogs of the enzyme. Since the current findings indicate that PanAm inhibition is based on the stability of its product complex with the enzyme, it is possible that the 4'-deoxy-PanAms have a different kinetic inhibition mechanism. If so, one should exhibit caution when using the kinetic results obtained with 4'-deoxy-PanAms to interpret PanAm inhibition.

We are currently re-

evaluating the proposed kinetic model to determine if it can be modified to describe the 4'-deoxyPanAm results obtained previously, as well as the results for PanAm inhibition obtained here. The antistaphylococcal susceptibility results highlight two important aspects related to PanAm inhibition: first, that regardless of the identity of the N-substituent, the compounds all target Pan-related processes as their inhibitory potency is highly sensitive to the amount of Pan present in the growth medium. Second, the potency of SaPanKII inhibition is not a good predictor of PanAm growth inhibition, as the two new PanAms discovered here are still less potent antistaphylococcal agents than N7-Pan (7) in spite of showing improved PanK inhibition. Although the mechanism of Pan/PanAm transport across the S. aureus membrane is not well understood, it is possible that the introduction of the polar ether group in MeO-N5-PanAm (11) and N-Pip-PanAm (12) may hinder membrane permeation. Consequently, this feature will need to be addressed if the PanAms as a class are to be moved forward as antistaphylococcals with potential for clinical use. However, the results presented here also offers direction for the development of new SaPanKII inhibitors that deviate from the PanAm scaffold through the exploitation of the important ligand-binding interactions identified in this work.

Hughes et al.

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METHODS General materials and methods. All PanAms were prepared synthetically and their purity confirmed by 1H NMR analysis, as described previously.39 The PanAms were dissolved in 50% acetonitrile– water solution to yield stock solutions at a concentration of 50–200 mM and assays were performed with the final acetonitrile concentration never exceeding 3% (v/v). General chemicals, reagents and media were purchased from Sigma-Aldrich or Merck Chemicals (Darmstadt, Germany) and were of the highest purity. Solvents used for reactions were Chromasolv HPLC grade solvents (SigmaAldrich) and the hexanes, dichloromethane (DCM) and ethyl acetate (EtOAc) used for purification were purchased from Merck Chemicals. Dry N,N-dimethylformamide (DMF) was prepared by shaking up over potassium hydroxide, distilled under reduced pressure and a nitrogen atmosphere, and finally stored over 4 Å molecular sieves in the dark. Dry tetrahydrofuran was distilled from sodium under a nitrogen atmosphere. The S. aureus RN4220 strain was a kind gift from L. M. T. Dicks at the Department of Microbiology (Stellenbosch University).

PK and LDH used in the kinetic assays were obtained

from Roche (Basel, Switzerland). All 1H and 13C NMR spectra were obtained using a 300-MHz Varian VNMRS (75 MHz for 13

C), 400-MHz Varian Unity Inova (100 MHz for

for

13

13

C) or 600 MHz Varian Unity Inova (150 MHz

C) instruments (Varian Inc., Palo Alto, CA, USA) at the Central Analytical Facility (CAF) of

Stellenbosch University. All chemical shifts (δ) were recorded using the residual solvent peak and reported in p.p.m. All high resolution mass spectrometry (HRMS) was performed on a Waters API Q-TOF Ultima spectrometer (Waters, Milford, MA, USA) at the MS unit of CAF. All OD600 measurements and kinetic studies were performed using a Thermo Varioskan spectrophotometer (Thermo Scientific, Bremen, Germany). Inhibition studies were performed in Greiner Bio-One Cellstar flat-bottomed 96-well suspension plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Kinetic studies were performed in Greiner Bio-One polystyrene flat-bottomed 96-well plates.

Synthesis of PanAms (Scheme S1). N-Heptylpantothenamide (N7-Pan, 7). Calcium pantothenate (1.00 g, 4.15 mmol) was exchanged to the free acid by dissolving the salt in H2O (15 mL) and passing the solution through a column (1.50 cm diameter) of pre-washed Amberlite IR-120 (H+form) ion exchange resin (1.10 g). The free carboxylic acid was eluted with H2O (1 × 15 mL),

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followed by lyophilization of the collected column eluate to afford pantothenic acid as a clear oil (860 mg, 3.92 mmol). Heptylamine (698 µL, 4.71 mmol) and diphenyl phosphoryl azide (DPPA) (1.02 mL, 4.71 mmol) were added to a solution of pantothenic acid (860 mg, 3.92 mmol) in anhydrous DMF (5 mL) at rt under an inert atmosphere. After cooling the mixture to 0 °C, triethylamine (Et3N) (656 µL, 4.71 mmol) was added. The reaction mixture was stirred for an additional 2h at 0 °C and left to stir overnight at rt. DMF was removed in vacuo and Amberlite IR400 (OH- form) resin was added (5.00 g). The reaction mixture was filtered and lyophilized before purification by FCC (10% methanol (MeOH) in DCM) afforded the desired amide (580 mg, 47%) as a white solid. Rf = 0.27 (FCC conditions). δH (300 MHz; CDCl3; 25 °C) 0.85 (3H, t, J = 7.0 Hz, CH3), 0.92 (3H, s, -CH3), 1.02 (3H, s, -CH3), 1.25-1.34 (8H, m, -(CH2)4-), 1.46 (2H, t, J = 6.5 Hz, CH2-), 2.40 (2H, t, J = 6.0 Hz, -CH2-), 3.19 (2H, q, J = 7.0 Hz, -CH2-), 3.49 (2H, s, -CH2-), 3.53-3.59 (2H, m, -CH2-), 3.98 (1H, s, -CH-), 5.79 (1H, br s, -NH-) and 7.33 (1H, br t, J = 6.2 Hz, -NH-). OH protons not observed. 1H NMR data are consistent with those previously reported.39 N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)pantothenamide (N-HoPip-PanAm, 8). To a solution of S-phenyl thiopantothenate40 (140 mg, 0.449 mmol) in acetonitrile (CH3CN) (5 mL) at rt was added N-homopiperonylamine (89.1 mg, 0.539 mmol). The reaction mixture was stirred overnight at 35 °C. The reaction mixture was concentrated in vacuo before purification by FCC (5% to 10% MeOH in DCM) afforded the desired amide (62.0 mg, 38%) as an orange oil. Rf = 0.13 (5% MeOH in DCM). δH (400 MHz; CDCl3; 25°C). 0.91 (3H, s, -CH3), 0.99 (3H, s, -CH3), 2.37 (2H, t, J = 5.9 Hz, -CH2-), 2.69 (2H, t, J = 7.0 Hz, -CH2-), 3.40-3.47 (4H, m, -(CH2)2-), 3.50-3.55 (2H, m, -CH2-), 3.99 (1H, s, CH-), 5.92 (2H, s, -CH2-), 6.14 (1H, br t, J = 5.5 Hz, -NH-), 6.60 (1H, dd, J = 1.6, 7.8 Hz, arom), 6.66 (1H, d, J = 1.6 Hz, arom), 6.72 (1H, d, J = 7.8 Hz, arom) and 7.41 (1H, br t, J = 5.5 Hz, -NH-). OH protons were not observed. 1H NMR data are consistent with those previously reported.32 N-(3-Methoxyphenethyl)pantothenamide (N-3-MeO-PE-PanAm, 9). Calcium pantothenate (1.00 g, 4.15 mmol) was exchanged to the free acid by dissolving the salt in H2O (15 mL) and passing the solution through a column (1.50 cm diameter) of pre-washed Amberlite IR-120 (H+form) ion exchange resin (1.10 g). The free carboxylic acid was eluted with H2O (1 × 15 mL), followed by lyophilization of the collected column eluate to afford pantothenic acid as a clear oil (840 mg, 3.83 mmol). 3-Methoxy-phenethylamine (615 µL, 4.22 mmol) and diethyl phosphoryl cyanide (DEPC) (611 µL, 4.22 mmol) were added to a solution of pantothenic acid (840 mg, 3.83 mmol) in anhydrous DMF (5 mL) at room temperature under an inert atmosphere. The reaction mixture was cooled to 0 °C before Et3N (1.12 mL, 8.04 mmol) was added. The reaction mixture was Hughes et al.

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stirred for 2 h at 0 °C and left to stir overnight at rt. EtOAc (50 mL) was added and the organic layer was washed with 5% aqueous citric acid (3 × 10 mL), 1 M aqueous NaHCO3 (2 × 10 mL) and sat. aqueous NaCl (1 × 10 mL). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo before purification by FCC (10% MeOH in DCM) afforded the desired amide (0.470 g, 35%) as a yellow oil. Rf = 0.30 (FCC conditions). δH (600 MHz; CDCl3; 25 °C). 0.91 (3H, s, -CH3), 0.98 (3H, s, -CH3), 2.37 (2H, t, J = 6.2 Hz, -CH2-), 2.76 (2H, t, J = 6.7 Hz, -CH2-), 3.44-4.47 (2H, m, -CH2-), 3.47-3.53 (4H, m, -(CH2)2-), 3.79 (3H, s, -CH3), 3.97 (1H, s, -CH-), 6.23 (1H, br t, J = 5.6 Hz, -NH-), 6.73 (1H, s, arom), 6.76 (2H, dd, J = 2.1, 7.9 Hz, arom), 7.20 (2H, t, J = 7.9 Hz, arom) and 7.43 (1H, br t, J = 5.9 Hz, -NH-). OH protons not observed. δC (150 MHz; CDCl3; 25°C) 18.8, 18.8, 37.9, 38.1, 38.4, 42.0, 45.2, 57.8, 73.4, 80.0, 114.3, 117,3, 123.7, 132.3, 142.9, 162.4, 174.1 and 176.4. (HRMS) [M+H]+ 353.2079 (Calculated [C18H29N2O5]+ = 353.2076). N-Phenethylpantothenamide (N-PE-PanAm, 10). Prepared as for 9 from pantothenic acid (860 mg, 3.92 mmol), phenethylamine (543 µL, 4.31 mmol), DEPC (654 µL, 4.31 mmol) and Et3N (1.15 mL, 8.23 mmol). Purification by FCC (5% MeOH in DCM) afforded the desired amide (554 mg, 44%) as a colourless oil. Rf = 0.29 (FCC conditions). δH (400 MHz; CDCl3; 25 °C) 0.92 (3H, s, CH3), 1.00 (3H, s, -CH3), 2.37 (2H, t, J = 6.1 Hz, -CH2-), 2.78 (2H, t, J = 7.2 Hz, -CH2-), 3.47 (2H, s, -CH2-), 3.50-3.55 (4H, m, -(CH2)2-), 3.97 (1H, s, -CH-), 6.10 (1H, br t, J = 5.5 Hz, -NH-), 7.17 (2H, d, J = 6.6 Hz, arom), 7.21 (1H, t, J = 7.4 Hz, arom), 7.29 (2H, t, J = 7.2 Hz, arom) and 7.40 (1H, br t, J = 6.1 Hz, -NH-). OH protons were not observed. 1H NMR data are consistent with those previously reported.32 N-(5-Methoxypentyl)pantothenamide (MeO-N5-PanAm, 11).

To a solution of S-ethyl

thiopantothenate40 (150 mg, 0.570 mmol) in CH3CN (5 mL) at rt was added 5-methoxypentan-1amine (see below) (167 mg, 1.43 mmol). The reaction mixture was stirred for 96 h at 45 °C. The reaction mixture was concentrated in vacuo before purification by FCC (10% MeOH in DCM) afforded the desired amide (172 mg, 96%) as an yellow oil. Rf = 0.18 (FCC conditions). δH (400 MHz; CDCl3; 25 °C). 0.89 (3H, s, -CH3), 0.97 (3H, s, -CH3), 1.31-1.39 (2H, m, -CH2-), 1.46-1.59 (4H, m, -(CH2)2-), 2.40 (2H, t, J = 5.5 Hz, -CH2-), 3.13-3.27 (2H, m, -CH2-), 3.31 (3H, s, -CH3), 3.34 (2H, t, J = 6.6 Hz, -CH2-), 3.45 (2H, s, -CH2-), 3.50-3.55 (2H, m, -CH2-), 3.95 (1H, apparent d, J = 4.7 Hz, -CH-), 6.45 (1H, br t, J = 5.5 Hz, -NH-) and 7.47 (1H, br t, J = 6.2 Hz, -NH-). OH protons not observed. δC (100 MHz; CDCl3; 25°C) 22.9, 24.0, 24.1, 26.2, 31.8, 37.9, 38.5, 42.0, 42.1, 61.2, 73.4, 75.3, 79.9 174.0 and 176.6. (HRMS) [M+H]+ 319.2240 (Calculated [C15H31N2O5]+ = 319.2233). Hughes et al.

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N-(benzo[d][1,3]dioxol-5-ylmethyl)pantothenamide (N-Pip-PanAm, 12). Prepared as for 9 from pantothenic acid (420 mg, 1.92 mmol), piperonylamine (263 µL, 2.11 mmol) and DEPC (320 µL, 2.11 mmol) in anhydrous DMF (3 mL) at rt under an inert atmosphere. The reaction mixture was cooled to 0 °C before Et3N (560 µL, 4.02 mmol) was added. The reaction mixture was stirred for 2 h at 0 °C and left to stir overnight at rt. EtOAc (15 mL) was added and the organic layer was washed with 5% aqueous citric acid (3 × 5 mL), 1 M aqueous NaHCO3 (2 × 5 mL) and sat. aqueous NaCl (1 × 5 mL). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo before purification by FCC (10% MeOH in DCM) afforded the desired amide (257 mg, 38%) as a yellow oil. Rf = 0.14 (FCC conditions). δH (300 MHz; CDCl3; 25 °C). 0.90 (3H, s, -CH3), 1.00 (3H, s, CH3), 2.44 (2H, t, J = 6.0 Hz, -CH2-), 3.47 (2H, s, -CH2-), 3.55-3.61 (2H, m, -CH2-), 3.96 (1H, s, CH-), 4.24 (1H, dd, J = 5.9, 14.7 Hz, -CH2-), 4.33 (1H, dd, J = 5.9, 14.7 Hz, -CH2-), 5.94 (2H, s, CH2-), 6.20 (1H, br s, -NH-), 6.69-6.76 (3H, m, arom) and 7.63 (1H, br s, -NH-). OH protons were not observed. 1H NMR data are consistent with those previously reported.39 5-Methoxypentan-1-amine. An oven dried two-necked round bottom flask was allowed to cool to rt under an inert atmosphere and the flask was charged via syringe with dry THF (40 mL). Sodium hydride (80% dispersion in mineral oil) (419 mg, 14.7 mmol) was added with a brief opening of the gas adapter. A solution of benzyl 5-hydroxypentylcarbamate41 (2.00 g, 8.43 mmol) in THF was added via syringe carefully and the reaction mixture was stirred for an additional 10 min. The reaction mixture was cooled to 0 °C before iodomethane (525 µL, 8.43 mmol) was added dropwise via syringe over 10 min. The reaction mixture was allowed to warm up to rt and stirred for 1 h at rt. The reaction mixture was quenched with H2O (20 mL), concentrated in vacuo and the aqueous layer was extracted with EtOAc (3 × 35 mL). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo before purification by FCC (1:1 EtOAc: Hexanes) afforded the desired benzyl 5-methoxypentylcarbamate (1.06 g, 50%) as a yellow oil. Rf = 0.66 (FCC conditions). δH (400 MHz; CDCl3; 25 °C). 1.33-1.42 (2H, m, -CH2-), 1.48-1.63 (4H, m, -(CH2)2-), 3.16 (2H, q, J = 6.5 Hz, -CH2-), 3.32 (3H, s, -CH3), 3.60 (2H, t, J = 6.3 Hz, -CH2-), 4.80 (1H, br s, -NH-), 5.09 (2H, s, -CH2-) and 7.30-7.36 (5H, m, arom). δC (100 MHz; CDCl3; 25 °C) 23.6, 29.5, 30.0, 41.2, 58.8, 66.8, 72.8, 128.3, 128.6, 128.7, 136.9 and 156.6. (HRMS) [M+H]+ 252.1603 (Calculated [C14H22NO3]+ = 252.1600). To a solution of benzyl 5-methoxypentylcarbamate (2.04 g, 8.12 mmol) in MeOH (65 mL) at rt was added 10% palladium on carbon (Pd/C) (346 mg, 3.25 mmol). The reaction atmosphere was filled with H2 gas and the reaction mixture was stirred overnight at rt. The reaction mixture was Hughes et al.

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filtered and concentrated in vacuo to give 5-methoxypentan-1-amine (879 mg, 92%) as a clear oil. Rf = product on baseline (10% MeOH in DCM). δH (400 MHz; CDCl3; 25 °C) 1.35-1.42 (2H, m, -CH2), 1.47-1.54 (2H, m, -CH2-), 1.55-1.62 (2H, m, -CH2-), 2.66 (3H, s, -CH3), 2.70 (2H, t, J = 7.0 Hz, CH2-) and 5.55 (2H, br s, -NH2). 1H NMR data are consistent with those previously reported.42

Protein expression and purification for structural analysis. The gene encoding for SaPanKII was cloned into expression vector pET28a containing a C-terminal His6-tag, and the resulting vector was transformed into E. coli BL-21(DE3) competent cells. Successful transformants were grown in LB media (Sigma) for 16 h at 37 °C, and then transferred to Terrific Broth (Sigma). Growth at 37 °C continued until the OD600 of the culture reached ~4, after which the temperature was lowered to 18 °C and protein expression was induced by the addition of isopropyl β-D-thiogalactopyranoside (IPTG). Eighteen hours post-induction, the cells were harvested by centrifugation and resuspended in binding buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM TCEP, 5% Glycerol, 10 mM imidazole) supplemented with 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM benzamidine and 500 units of benzonase nuclease.

The cells were lysed by sonication and the lysate was clarified by

centrifugation. The supernatant was loaded onto nickel-nitriloacetic acid resin beads, washed with binding buffer containing 30 mM imidazole, and bound protein was eluted with 10 mL of binding buffer containing 300 mM imidazole. The eluate was desalted using a HiPrep 26/10 desalting column (GE Healthcare) equilibrated with a salt-free buffer (Buffer A: 20 mM HEPES, pH 7.5, 1 mM TCEP, 5% Glycerol). Fractions containing protein were pooled and loaded onto a HiTrap Q HP column (GE Healthcare) equilibrated using Buffer A. Bound protein was eluted using a 0–1 M NaCl gradient. Highest purity fractions were then pooled and loaded onto a HiLoad Superdex TM 75 column (GE Healthcare) equilibrated with gel filtration buffer (20 mM Tris HCl, pH 8.0, 200 mM NaCl and 10 mM dithiothreitol). Highest purity fractions were concentrated with Amicon Ultra-15 centrifugal filters (Millipore) to 20–30 mg/mL, aliquoted and frozen at -80 °C until further use. Crystallization and Data collection. Crystallization trials were carried out using the sitting drop vapor-diffusion method and both in-house and commercially available (Hampton research) screening kits. Trials were conducted using 0.5 µL protein + 0.5 µL buffer per drop, and trays were kept at 18 °C. Prior to crystallization, purified SaPanKII was incubated overnight at 4 °C in the presence of PanAms, MgCl2 and ATP (10 mM final concentration). Initial poor quality crystals of SaPanKII with Hughes et al.

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N5-Pan (6) and ATP were obtained in 2.5 M (NH4)2SO4 and 0.1 M bis-tris propane, pH 7.0. The addition of 1.1% w/v cyclohexylethanoyl-N-hydroxyethylglucamide produced diffraction quality crystals. Crystals of SaPanKII with N7-Pan 7 and ATP were obtained using 30% PEG4K, 0.2 M MgCl2, 0.1 M Tris, pH 8.5. High quality crystals of SaPanKII, ATP and N-Pip-PanAm (12) were obtained from a reservoir solution containing 25% PEG3350, 0.2 M MgCl2, 0.1 M Tris, pH 8.5 by utilizing in situ proteolysis with 1:500 elastase.43 Crystals were cryoprotected in 50% paratone:50% mineral oil, flash-frozen and stored in liquid nitrogen until data collection. Prior to data collection, crystals of SaPanKII with N5-Pan were separated under paratone oil from a stack that was originally frozen. Data were collected on a rotating copper anode source at the Structural Genomics Consortium (SGC, Toronto), the Advanced Photon Source (Argonne National Laboratory; Argonne, IL) or the Canadian Light Source Inc (CLSI, University of Saskatchewan; Saskatoon, Canada). N7Pan (7), MeO-N5-PanAm (11) and N-Pip-PanAm (12) data were processed using XDS/XSCALE44 and SCALA,45 while N5-Pan (6) data were processed using XDS/XSCALE44 and XPREP (Bruker AXS Inc.). Structures were solved by molecular replacement using PHASER46 from the CCP4 crystallographic suite47 and a previously solved SaPanKII structure (PDB ID: 2EWS20) as a search model. The models underwent several rounds of model building and refinement with COOT48 and REFMAC549, respectively. N5-Pan (6) and N7-Pan (7) geometry restraints for refinement were prepared with PRODRG50 and MOGUL51 based on ligand coordinates from PDB entry 3SMS. NPip-PanAm (12) and MeO-N5-PanAm (11) restraints were generated using eLBOW52 and PRODRG. Model geometry was validated using MOLPROBITY.53 Coordinates for all four SaPanKII structures have been deposited in the Protein Data Bank with accession codes 4M7Y (N5-Pan, 6), 4M7X (N7Pan, 7), 5JIC (MeO-N5-PanAm, 11) and 5ELZ (N-Pip-PanAm, 12). Data collection and refinement statistics are shown in Table 1. Figures were generated using the PyMOL Molecular Graphics System (Version 1.5.0.4 Schrödinger, LLC).

Sequence alignments were performed using the

ESPript 3.0 server (http://espript.ibcp.fr).54 The crystals of N7-Pan-, MeO-N5-PanAm- and N-Pip-PanAm-bound SaPanKII belong to space group C2221 with one molecule in the asymmetric unit (ASU). A crystallographic rotation generates a dimer consistent with the previous report,20 and the final models were refined to 1.42, 1.40, and 1.80 Å resolution, respectively. The N5-Pan-bound SaPanKII crystal belongs to the space group C2, and there are two molecules in the ASU. These two molecules represent a biological dimer of SaPanKII. The final model of N5-Pan-bound SaPanKII was refined to 1.80 Å (Fig. 2a). For Hughes et al.

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each structure the protein was incubated with PanAm, ATP and MgCl2, but the electron density maps were consistent with ADP, Mg2+ and phosphorylated PanAm.

SaPanKII activity assays. SaPanKII activity with the various PanAms was determined using a continuous spectrophotometric assay that coupled the production of ADP to the consumption of NADH (monitored by following the decrease in A340) by using pyruvate kinase (PK) and lactate dehydrogenase (LDH), and were performed as described previously.25 Each 300 µL reaction mixture contained 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 20 mM KCl, 1.5 mM ATP, 0.5 mM NADH, 0.5 mM phosphoenolpyruvate, 3 units of PK, 3 units of LDH and 1.5 µg of SaPanKII (prepared as described previously25).

The reaction was initiated by the addition of a particular PanAm

(concentrations ranged between 3.125 and 200 µM) and was monitored for 5 min at 25 °C. An extinction coefficient of 6220 M-1·cm-1 was used for NADH. Initial velocities at each substrate concentration (tested in triplicate) were determined from the raw kinetic data as described previously.25 Briefly, initial velocities were calculated by linear regression of the readings made in the period from 10 to 60 s after the reaction was started). For each experiment, the triplicate readings made for each substrate concentration were averaged and plotted with the standard deviation to give an activity profile. Kinetic parameters reported in Table S1 were determined for each experiment by fitting a simple Hill-type equation (Eq. S1) to all the data simultaneously; the reported values are the mean values of the parameters determined from all the individual independent experiments (number indicated in Table S1), and are given with errors that indicate the range/2 or SEM as appropriate.

SaPanKII inhibition assays. IC50 values for inhibition of SaPanKII activity by the various PanAms was determined using the assay described above. A constant substrate concentration of 25 µM Pan was used; PanAm concentrations were varied between 0.05–3.125 µM (for PanAm 11), 0.1–6.25 µM (for PanAm 12) or 0.8–50 µM (for PanAms 7, 8, 9 & 10). Initial rates were determined (in triplicate) and normalized to the rate of the reaction without inhibitor. A concentration–response curve for each experiment was plotted using the average activity of the triplicate readings. IC50 values for each experiment were determined by fitting Eq. S2 (setting the y0 and a parameters to 0 and 100 respectively) to all the data points simultaneously. The values reported in Table S1 and shown in Fig. 3a are the mean values of the parameters determined from all the individual independent experiments (number indicated in Table S1), and are given with errors that indicate the range/2 or SEM as appropriate. Hughes et al.

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Antistaphylococcal susceptibility assays. In minimal media. The PanAm-mediated inhibition of S. aureus grown in minimal media was determined as described previously.25 Briefly, a starter culture of S. aureus RN4220 in 1% tryptone was inoculated with four separate colonies grown on LB agar plates. The starter culture was grown to exponential phase and then diluted 10-fold into the minimal media. A 10 µL aliquot of the diluted cell suspension was used to inoculate each well of a 96-well flat-bottomed plate containing 100 µL of minimal medium constituted as previously described25) supplemented with the specific compound of interest (diluted from 50–200 mM stock solutions prepared in 50% aqueous CH3CN to aid solubility). Final concentrations of compounds were in the range 0.0078–8 µM depending on the potency of the PanAm. The plates were incubated at 37 °C for 24 h before the cell densities were measured (OD600). The extent of growth in each well was determined by normalizing the OD600 values relative to those of the negative control (containing 3% CH3CN instead of pantothenamide), which was taken as 100% bacterial cell growth.

Each

compound was tested in triplicate and all experiments were repeated at least once after the initial experiment. The reported MIC values were determined by concentration–response analysis of the individual experiments, and are lowest concentration at which the indicated % inhibition (relative to an uninhibited control) was observed. In 1% tryptone. The PanAm-mediated inhibition of S. aureus grown in 1% tryptone was determined as described previously.25 Briefly, a starter culture of S. aureus RN4220 in 1% tryptone was prepared by inoculation with four separate colonies grown on LB agar plates. The starter culture was grown to exponential phase and then diluted 10 000-fold in the same medium. A 10 µL aliquot of the diluted cell suspension was used to inoculate each well of a 96-well plate containing 100 µL of 1% tryptone broth supplemented with the specific compound of interest (diluted from 50–200 mM stock solutions prepared in 50% aqueous CH3CN to aid solubility). Final concentrations of compounds were in the range 0.016–50 µM depending on the potency of the PanAm. The plates were incubated at 37 °C for 20 h before the cell densities were measured (OD600) and analyzed further as described above. In Mueller-Hinton broth. Susceptibility assays in compliance with the guidelines of the Clinical and Laboratory Standards Institute55 were determined using a broth macrodilution assay as described elsewhere.56 Briefly, S. aureus (ATCC® 29213) cells (~5 x 105 cfu/mL) were grown in Mueller-Hinton broth (MHB; Gibco®) containing increasing concentrations of test compound. After

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overnight growth the OD600 was determined for each condition, with the MIC defined as the lowest concentration yielding no measurable absorbance.

ABBREVIATIONS ACP, acyl carrier protein ADP, adenosine diphosphate ATP, adenosine triphosphate BaPanKIII, Bacillus anthracis type III pantothenate kinase CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate EcPanKI, Escherichia coli type I pantothenate kinase IPTG, isopropyl β-D-thiogalactopyranoside KpPanKI, Klebsiella pneumoniae type I pantothenate kinase MHB, Mueller-Hinton Broth MIC, minimal inhibitory concentration MDR, Multidrug-resistant MeO-N5-PanAm, N-(5-methoxypentyl)pantothenamide MRSA, Methicillin-resistant Staphylococcus aureus N-3-MeO-PE-PanAm, N-[(3-methoxy)-phenethyl]pantothenamide N5-Pan, N-pentylpantothenamide N7-Pan, N-heptylpantothenamide NADH, nicotinamide adenine dinucleotide N-HoPip-PanAm, N-homopiperonylpantothenamide N-PE-PanAm, N-phenethylpantothenamide N-Pip-PanAm, N-piperonylpantothenamide Pan, pantothenate P-Pan, 4'-phosphopantothenic acid PanAm, pantothenamide PanK, pantothenate kinase PantSH, pantetheine PaPanKIII, Pseudomonas aeruginosa type III pantothenate kinase PCP, peptidyl carrier protein PK/LDH, pyruvate kinase / lactate dehydrogenase Hughes et al.

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PMSF, phenylmethanesulfonylfluoride PPAT, phosphopantetheine adenylyltransferase SaPanKII, Staphylococcus aureus type II pantothenate kinase SAR, structure activity relationship SEM, standard error of the mean TCEP, tris(2-carboxyethyl)phosphine

ASSOCIATED CONTENT Supporting Information This information is available free of charge via the Internet at http://pubs.acs.org/. Additional Figure S1, Table S1, Scheme S1, Equations S1 and S2, and extended discussion. (PDF)

AUTHOR INFORMATION Corresponding Authors *(Erick Strauss) E-mail: [email protected]. *(Hee-Won Park) E-mail: [email protected]. Author Contributions ○

S.H. and L.B. contributed equally.

Author Contributions L.B.: Chemical synthesis of PanAms, enzyme activity and inhibition assays, bacterial cell inhibition tests. S.J.H: Structure determination, enzyme activity assays, bacterial cell inhibition tests. K.M.: Expression, purification, crystallization, bacterial cell inhibition tests. W.T.: Structure determination of N5-Pan 6 and N7-Pan 7 complexes. T.A.: Expression, purification, crystallization. B.S.H.: Structure determination and bacterial cell inhibition tests. A.A.-H.: Enzyme activity and inhibition assays. D.S.: Chemical synthesis of PanAms. M.V.: Designed and directed enzyme activity and inhibition assays. E.S., H.-W.P.: Conceptualized the project, directed the study and wrote the paper (with input from all authors). Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Mehnaz Pervin and Dr. Beong Ou Lim (Department of Applied Biochemistry, Konkuk University, Chungju, Korea) for providing resources, facilities and assistance in carrying out the MIC assays in Mueller-Hinton broth. This work was supported by the Defense Medical Research and Development Program FY10 Basic Research Award DM102976 (H.-W.P.), Louisiana board of Regents grants LEQSF(2015-18)-RD-A-27 and LEQSF-EPS(2015)-PFUND-417 (H.-W.P.) and NRF CPRR Grant 93430 (E.S.). L.B. is a recipient of an NRF Scarce Skills Scholarship. Research described in this paper was in part performed beamline 08ID-1 at the Canadian Light Source and the Structural Biology Center beamline 19-ID at the Advanced Photon Source at Argonne National Laboratory. The 08ID-1 beamline is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. Research described in this paper was performed in part at the Structural Genomics Consortium (SGC). The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada through the Ontario Genomics Institute [OGI-055], GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust [092809/Z/10/Z].

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(23) Balibar, C. J., Hollis-Symynkywicz, M. F., and Tao, J. (2011) Pantethine Rescues Phosphopantothenoylcysteine Synthetase and Phosphopantothenoylcysteine Decarboxylase Deficiency in Escherichia coli but Not in Pseudomonas aeruginosa, J. Bacteriol. 193, 33043312. (24) Leonardi, R., Chohnan, S., Zhang, Y. M., Virga, K. G., Lee, R. E., Rock, C. O., and Jackowski, S. (2005) A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A, J. Biol. Chem. 280, 3314-3322. (25) de Villiers, M., Barnard, L., Koekemoer, L., Snoep, J. L., and Strauss, E. (2014) Variation in Pantothenate Kinase Type Determines Pantothenamide Mode of Action and Impacts on Coenzyme A Salvage Biosynthesis, FEBS J. 281, 4731-4753. (26) Clifton, G., Bryant, S. R., and Skinner, C. G. (1970) N'-(substituted) pantothenamides, antimetabolites of pantothenic acid, Arch. Biochem. Biophys. 137, 523-528. (27) Saliba, Kevin J., and Spry, C. (2014) Exploiting the coenzyme A biosynthesis pathway for the identification of new antimalarial agents: the case for pantothenamides, Biochemical Society Transactions 42, 1087-1093. (28) de Villiers, M., Macuamule, C., Spry, C., Hyun, Y. M., Strauss, E., and Saliba, K. J. (2013) Structural modification of pantothenamides counteracts degradation by pantetheinase and improves antiplasmodial activity, ACS Med. Chem. Lett. 4, 784-789. (29) Jansen, P. A., Hermkens, P. H., Zeeuwen, P. L., Botman, P. N., Blaauw, R. H., Burghout, P., van Galen, P. M., Mouton, J. W., Rutjes, F. P., and Schalkwijk, J. (2013) Combination of pantothenamides with vanin inhibitors as a novel antibiotic strategy against gram-positive bacteria, Antimicrob. Agents Chemother. 57, 4794-4800. (30) Spry, C., Macuamule, C., Lin, Z., Virga, K. G., Lee, R. E., Strauss, E., and Saliba, K. J. (2013) Pantothenamides are potent, on-target inhibitors of Plasmodium falciparum growth when serum pantetheinase is inactivated, PLoS One 8, e54974. (31) Macuamule, C. J., Tjhin, E. T., Jana, C. E., Barnard, L., Koekemoer, L., de Villiers, M., Saliba, K. J., and Strauss, E. (2015) A Pantetheinase-Resistant Pantothenamide with Potent, OnTarget, and Selective Antiplasmodial Activity, Antimicrob. Agents Chemother. 59, 36663668. (32) Hughes, S. J., Antoshchenko, T., Kim, K. P., Smil, D., and Park, H. W. (2014) Structural characterization of a new N-substituted pantothenamide bound to pantothenate kinases from Klebsiella pneumoniae and Staphylococcus aureus, Proteins 82, 1542-1548. (33) Strauss, E., and Begley, T. P. (2002) The antibiotic activity of N-pentylpantothenamide results from its conversion to ethyldethia-coenzyme a, a coenzyme a antimetabolite, J. Biol. Chem. 277, 48205-48209. (34) Zhang, Y. M., Frank, M. W., Virga, K. G., Lee, R. E., Rock, C. O., and Jackowski, S. (2004) Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites, J. Biol. Chem. 279, 50969-50975. (35) Choudhry, A. E., Mandichak, T. L., Broskey, J. P., Egolf, R. W., Kinsland, C., Begley, T. P., Seefeld, M. A., Ku, T. W., Brown, J. R., Zalacain, M., and Ratnam, K. (2003) Inhibitors of pantothenate kinase: novel antibiotics for staphylococcal infections, Antimicrob. Agents Chemother. 47, 2051-2055. (36) Virga, K. G., Zhang, Y. M., Leonardi, R., Ivey, R. A., Hevener, K., Park, H. W., Jackowski, S., Rock, C. O., and Lee, R. E. (2006) Structure-activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors, Bioorg. Med. Chem. 14, 1007-1020. (37) Leonardi, R., Zhang, Y.-M., Yun, M.-K., Zhou, R., Zeng, F.-Y., Lin, W., Cui, J., Chen, T., Rock, C. O., White, S. W., and Jackowski, S. (2010) Modulation of Pantothenate Kinase 3

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TABLES Table 1. Data collection and refinement statistics for SaPanKII structures Ligand PDB ID Data Collection Beamline

Wavelength (Å) Resolution (Å)a

N5-Pan 4M7Y

N7-Pan 4M7X

MeO-N5-PanAm 5JIC

N-Pip-PanAm 5ELZ

Rigaku FR-E

19-ID Advanced Photon Source (APS) 0.97918 36.8 - 1.42 (1.50 1.42) C2221 1 a=56.9, b=136.5, c=73.6 515456 (56576) 53304 (7569) 98.1 (96.5) 19.6 9.7 (7.5) 8.6 (73.6) 14.1 (2.4)

08ID-1 Canadian Light Source Inc. (CLSI) 0.97949 43.0 - 1.40 (1.43 1.40) C2221 1 a=57.4, b=136.8, c=73.9 532843 (80458) 57601 (8318) 99.9 (99.9) 16.7 9.3 (9.7) 8.7 (52.9) 13.0 (3.8)

Rigaku FR-E

1.5418 30.0 - 1.80 (1.85 1.80) C2 2 a=142.0, b=56.1, c=68.4, β=101.2 345634 (25407) 48878 (3589) 99.4 (99.0) 24.9 7.1 (7.1) 7.9 (95.0) 14.9 (2.0)

1.5418 28.5 - 1.80 (1.90 - 1.80) C2221 1 a=57.0, b=136.8, c=73.9 188546 (26323) 27062 (3880) 99.9 (99.2) 21.5 7.0 (6.8) 5.8 (49.8) 23.1 (4.0)

Space Group No. of molecules in ASU Unit cell parameters (Å, degrees) No. of measured reflections No. of unique reflections Completeness (%) Wilson B-Factor (Å2) Friedel Redundancy Rmerge (%)b Average I/σ Refinement Resolution (Å) 26.5 - 1.80 35.0 - 1.42 43.0 - 1.40 28.0 - 1.80 Rwork/Rfree (%)c 17.6/22.0 17.1/19.4 14.2/17.4 16.9 / 20.7 No. of atoms Protein 4061 2131 2240 2134 Ligand/ion 137 60 54 57 Water 194 147 328 275 Average B-factors (Å2) Protein 33.0 24.3 19.9 25.0 Ligand/ion 30.1 22.4 14.8 16.6 Water 33.3 30.3 31.5 31.3 RMSD bond length (Å) 0.014 0.015 0.012 0.010 RMSD bond angle (degrees) 1.54 1.51 1.30 1.26 Ramachandran Analysis49 Favored (%) 97.3 97.6 97.9 97.8 Outliers (%) 1d 1e 0 0 a Numbers in parentheses are for the outer shell. b Rmerge = Σ[(I − )]/Σ(I), where I is the observed intensity and is the average intensity. c Rwork = Σ[|Fobs| − |Fcalc|]/Σ|Fobs|, where |Fobs| and |Fcalc| are magnitudes of observed and calculated structure factors respectively. Rfree was calculated as Rwork using approximately 5.0% of the data, which was set aside for an unbiased test of the progress of refinement. d K161 is located in a disallowed region through interaction with neighbouring residues and water molecules e The outlier D19 is due to density located on an exterior loop of SaPanKII

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Table 2. PanAm-mediated growth inhibition of S. aureus in several media.a Compound

MIC (µM) (minimal medium)

Nb

MIC (µM) (1% tryptone)

Nb

MIC (µM) (Mueller-Hinton)

Nb

N5-Pan 6

~1.5c

2

~18c

2

85

4

N7-Pan 7