Toward a Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies of N-Phenethyl-α-methyl-pantothenamide.

Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the ß-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure-activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2'R-diastereomer is far more potent than the 2R,2'R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2'R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2'R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.

Overcoming synthetic challenges in targeting coenzyme A biosynthesis with the antimicrobial natural product CJ-15,801.

The biosynthesis of the essential metabolic cofactor coenzyme A (CoA) has been receiving increasing attention as a new target that shows potential to counter the rising resistance to established antimicrobials. In particular, phosphopantothenoylcysteine synthetase (PPCS)-the second CoA biosynthesis enzyme that is found as part of the bifunctional CoaBC protein in bacteria, but is monofunctional in eukaryotes-has been validated as a target through extensive genetic knockdown studies in Mycobacterium tuberculosis. Moreover, it has been identified as the molecular target of the fungal natural product CJ-15,801 that shows selective activity against Staphylococcus aureus and the malaria parasite Plasmodium falciparum. As such, CJ-15,801 and 4'-phospho-CJ-15,801 (its metabolically active form) are excellent tool compounds for use in the development of new antimicrobial PPCS inhibitors. Unfortunately, further study and analysis of CJ-15,801 is currently being hampered by several unique challenges posed by its synthesis. In this study we describe how these challenges were overcome by using a robust palladium-catalyzed coupling to form the key N-acyl vinylogous carbamate moiety with retention of stereochemistry, and by extensive investigation of protecting groups suited to the labile functional group combinations contained in this molecule. We also demonstrate that using TBAF for deprotection causes undesired off-target effects related to the presence of residual tertiary ammonium salts. Finally, we provide a new method for the chemoenzymatic preparation of 4'-phospho-CJ-15,801 on multi-milligram scale, after showing that chemical synthesis of the molecule is not practical. Taken together, the results of this study advances our pursuit to discover new antimicrobials that specifically target CoA biosynthesis and/or utilization.

Probing the ligand preferences of the three types of bacterial pantothenate kinase.

Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4'-phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here, a series of pantothenate analogues were synthesized using convenient synthetic methodology. The compounds were exploited as small organic probes to compare the ligand preferences of the three different types of bacterial PanK. Overall, several new inhibitors and substrates were identified for each type of PanK.

Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

Developing Pantetheinase-Resistant Pantothenamide Antibacterials: Structural Modification Impacts on PanK Interaction and Mode of Action.

Pantothenamides (PanAms) are analogues of pantothenate, the biosynthetic precursor of coenzyme A (CoA), and show potent antimicrobial activity against several bacteria and the malaria parasite in vitro. However, pantetheinase enzymes that normally degrade pantetheine in human serum also act on the PanAms, thereby reducing their potency. In this study, we designed analogues of the known antibacterial PanAm N-heptylpantothenamide (N7-Pan) to be resistant to pantetheinase by using three complementary structural modification strategies. We show that, while two of these are effective in imparting resistance, the introduced modifications have an impact on the analogues' interaction with pantothenate kinase (PanK, the first CoA biosynthetic enzyme), which acts as a metabolic activator and/or target of the PanAms. This, in turn, directly affects their mode of action. Importantly, we discover that the phosphorylated version of N7-Pan shows pantetheinase resistance and antistaphylococcal activity, providing a lead for future studies in the ongoing search of PanAm analogues that show in vivo efficacy.

Antiplasmodial Mode of Action of Pantothenamides: Pantothenate Kinase Serves as a Metabolic Activator Not as a Target.

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural ß-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites and PanAm phosphorylation by PfPanK present in parasite lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the ß-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.

Discovery of Potent Pantothenamide Inhibitors of Staphylococcus aureus Pantothenate Kinase through a Minimal SAR Study: Inhibition Is Due to Trapping of the Product.

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 those of other PanKs. To dissect the molecular interactions responsible for PanAm inhibitory activity, we conducted a mini SAR study in tandem with the cocrystallization 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 cocrystal structures showed that all of 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.

A pantetheinase-resistant pantothenamide with potent, on-target, and selective antiplasmodial activity.

Pantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC50], ∼20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of α-methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC50, 52 ± 6 nM), target specificity, and low toxicity.

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis.

N-substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well-studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide-induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA- and acyl carrier protein-dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanKI , PanKII and PanKIII ) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanKI enzymes accept them as substrates, the highly selective PanKIII s are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanKII ) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.