Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

LipE guided discovery of isopropylphenyl pyridazines as pantothenate kinase modulators.

Pantothenate kinase (PANK) is the critical regulator of intracellular levels of coenzyme A and has emerged as an attractive target for treating neurological and metabolic disorders. This report describes the optimization, synthesis, and full structure-activity relationships of a new chemical series of pantothenate competitive PANK inhibitors. Potent drug-like molecules were obtained by optimizing a high throughput screening hit, using lipophilic ligand efficiency (LipE) derived from human PANK3 IC50 values to guide ligand development. X-ray crystal structures of PANK3 with index inhibitors from the optimization were determined to rationalize the emerging structure activity relationships. The analysis revealed a key bidentate hydrogen bonding interaction between pyridazine and R306' as a major contributor to the LipE gain observed in the optimization. A tractable series of PANK3 modulators with nanomolar potency, excellent LipE values, desirable physicochemical properties, and a well-defined structural binding mode was produced from this study.

Metabolic activation of CaMKII by coenzyme A.

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

In Vivo and In Vitro Effects of a ClpP-Activating Antibiotic against Vancomycin-Resistant Enterococci.

Antibiotics with novel bactericidal mechanisms of action are urgently needed. The antibiotic acyldepsipeptide 4 (ADEP4) activates the ClpP protease and causes cells to self-digest. The effects of ADEP4 and ClpP activation have not been characterized sufficiently for the enterococci, which are important pathogens known for high levels of acquired and intrinsic antibiotic resistance. In the present study, ADEP4 was found to be potently active against both Enterococcus faecalis and Enterococcus faecium, with MIC90s of 0.016 µg/ml and 0.031 µg/ml, respectively. ClpP purified from E. faecium was found to bind ADEP4 in a surface plasmon resonance analysis, and ClpP activation by ADEP4 was demonstrated biochemically with a ß-casein digestion assay. In addition, E. faecium ClpP was crystallized in the presence of ADEP4, revealing ADEP4 binding to ClpP in the activated state. These results confirm that the anti-enterococcal activity of ADEP4 occurs through ClpP activation. In killing curve assays, ADEP4 was found to be bactericidal against stationary-phase vancomycin-resistant E. faecalis (VRE) strain V583, and resistance development was prevented when ADEP4 was combined with multiple classes of approved antibiotics. ADEP4 in combination with partnering antibiotics also eradicated mature VRE biofilms within 72 h of treatment. Biofilm killing with ADEP4 antibiotic combinations was superior to that with the clinically used combinations ampicillin-gentamicin and ampicillin-daptomycin. In a murine peritoneal septicemia model, ADEP4 alone was as effective as ampicillin. ADEP4 coadministered with ampicillin was significantly more effective than either drug alone. These data suggest that ClpP-activating antibiotics may be useful for treating enterococcal infections.

Discovery of an L-alanine ester prodrug of the Hsp90 inhibitor, MPC-3100.

Various types of Hsp90 inhibitors have been and continue to undergo clinical investigation. One development candidate is the purine-based, synthetic Hsp90 inhibitor 1 (MPC-3100), which successfully completed a phase I clinical study. However, further clinical development of 1 was hindered by poor solubility and consequent formulation issues and promoted development of a more water soluble prodrug. Towards this end, numerous pro-moieties were explored in vitro and in vivo. These studies resulted in identification of L-alanine ester mesylate, 2i (MPC-0767), which exhibited improved aqueous solubility, adequate chemical stability, and rapid bioconversion without the need for solubilizing excipients. Based on improved physical characteristics and favorable PK and PD profiles, 2i mesylate was selected for further development. A convergent, scalable, chromatography-free synthesis for 2i mesylate was developed to support further clinical evaluation.

Discovery of novel bacterial elongation condensing enzyme inhibitors by virtual screening.

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.

Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket.

The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

Pantothenate kinase activation relieves coenzyme A sequestration and improves mitochondrial function in mice with propionic acidemia.

Propionic acidemia (PA) is a rare autosomal-recessive metabolic disease that arises from mutations in propionyl-CoA (C3-CoA) carboxylase. Reduced enzyme activity slows C3-CoA metabolism, leading to an elevated plasma C3:C2-carnitine ratio, the hallmark biomarker of PA. The metabolic imbalances experienced in PA are however poorly defined. Here, we used a hypomorphic PA mouse model to demonstrate that C3-CoA accumulation in liver reduced non-esterified CoA (CoASH) and acetyl-CoA (C2-CoA). Tricarboxylic acid (TCA) cycle intermediates that are normally metabolized instead accumulated in urine, providing direct evidence for compromised mitochondrial function in PA. Pantothenate kinase (PanK) is known to catalyze the rate-controlling step in CoA biosynthesis, and its inhibition by C3-CoA prevents an increase in CoA biosynthesis to alleviate CoASH sequestration. PZ-3022 is an allosteric PanK activator that counteracts C3-CoA inhibition. PZ-3022 therapy increased hepatic CoASH and C2-CoA and decreased C3-CoA in the PA mouse model, leading to improved intracellular C3:C2-CoA and plasma C3:C2-carnitine ratios. Elevated urinary malate is a major component of the metabolic signature for TCA cycle dysfunction in the PA mouse, and the 80% reduction in urine malate by PZ-3022 therapy indicates the restoration of mitochondrial function. Thus, CoASH sequestration in PA leads to reduced TCA cycle activity that is relieved by PZ-3022, providing preclinical proof of concept for PanK activators as a therapy to attenuate the underlying mitochondrial defect in PA.

Ureadepsipeptides as ClpP Activators.

Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. Herein, we explore alteration of the potentially metabolically reactive α,ß unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.

Discovery of novel isoxazolines as anti-tuberculosis agents.

Nitrofuranyl isoxazolines with increased proteolytic stability over nitrofuranyl amides were designed and synthesized leading to discovery of several compounds with potent in vitro anti-tuberculosis activity. However, their in vivo activity was limited by high protein binding and poor distribution. Consequently, a series of non-nitrofuran containing isoxazolines were prepared to determine if the core had residual anti-tuberculosis activity. This led to the discovery of novel isoxazoline 12 as anti-tuberculosis agent with a MIC(90) value of 1.56microg/mL.

Synthesis and evaluation of cyclic secondary amine substituted phenyl and benzyl nitrofuranyl amides as novel antituberculosis agents.

In an ongoing effort to develop new and potent antituberculosis agents, a second-generation series of nitrofuranyl amides was synthesized on the basis of the lead compound 5-nitrofuran-2-carboxylic acid 3,4-dimethoxybenzylamide. The primary design consideration was to improve the solubility and consequently the bioavailability of the series by the addition of hydrophilic rings to the benzyl and phenyl B ring core. The synthesis of 27 cyclic, secondary amine substituted phenyl and benzyl nitrofuranyl amides is described and their activity against Mycobacterium tuberculosis reported. The series showed a strong structure-activity relationship as the benzyl nitrofuranyl amides were significantly more active than similarly substituted phenyl nitrofuranyl amides. Para-substituted benzyl piperazines showed the most antituberculosis activity. Compounds in the series were subsequently selected for bioavailability and in vivo testing. This study led to the successful discovery of novel compounds with increased antituberculosis activity in vitro and a better understanding of the requisite pharmacological properties to advance this class.

Synthesis and evaluation of nitrofuranylamides as novel antituberculosis agents.

In an effort to develop new and more potent therapies to treat tuberculosis, a library of compounds was screened for M. tuberculosis UDP-Gal mutase inhibition. Nitrofuranylamide 1 was identified as a hit in this screen, possessing good antituberculosis activity. This paper describes the synthesis and evaluation of an expanded set of nitrofuranylamides. We have discovered a number of nitrofuranylamides with submicromolar M. tuberculosis MIC values and acceptable therapeutic indexes. The MIC activity did not correlate with UDP-Gal mutase inhibition, suggesting an alternative primary cellular target was responsible for the antituberculosis activity. The compounds were only active against mycobacteria of the tuberculosis complex. On the basis of these results, four compounds were selected for in vivo testing in a mouse model of tuberculosis infection, and of these compounds one showed significant antituberculosis activity.

Discovery of (2S)-1-[4-(2-{6-amino-8-[(6-bromo-1,3-benzodioxol-5-yl)sulfanyl]-9H-purin-9-yl}ethyl)piperidin-1-yl]-2-hydroxypropan-1-one (MPC-3100), a purine-based Hsp90 inhibitor.

Modulation of Hsp90 (heat shock protein 90) function has been recognized as an attractive approach for cancer treatment, since many cancer cells depend on Hsp90 to maintain cellular homeostasis. This has spurred the search for small-molecule Hsp90 inhibitors. Here we describe our lead optimization studies centered on the purine-based Hsp90 inhibitor 28a containing a piperidine moiety at the purine N9 position. In this study, key SAR was established for the piperidine N-substituent and for the congeners of the 1,3-benzodioxole at C8. These efforts led to the identification of orally bioavailable 28g that exhibits good in vitro profiles and a characteristic molecular biomarker signature of Hsp90 inhibition both in vitro and in vivo. Favorable pharmacokinetic properties along with significant antitumor effects in multiple human cancer xenograft models led to the selection of 28g (MPC-3100) as a clinical candidate.

Synthesis, optimization and structure-activity relationships of 3,5-disubstituted isoxazolines as new anti-tuberculosis agents.

In the course of the development of a potent series of nitrofuranylamide anti-tuberculosis agents, we investigated if the exceptional activity resulted in part from the isoxazoline core and if it possessed any intrinsic anti-tuberculosis activity. This led to the discovery of an isoxazoline ester with appreciable anti-tuberculosis activity. In this study we explored the anti-tuberculosis structure-activity relationship of the isoxazoline ester compound through systematic modification of the 3,5-di-substituted isoxazoline core. Two approaches were used: (i) modification of the potentially metabolically labile ester functionality at the 3 position with acids, amines, amides, reverse amides, alcohols, hydrazides, and 1,3,4-oxadiazoles; (ii) substitution of the distal benzyl piperazine ring in the 5 position of the isoxazoline ring with piperazyl-ureas, piperazyl-carbamates, biaryl systems, piperidines and morpholine. Attempts to replace the ester group at C-3 position of isoxazoline with a variety of bioisosteric head groups led to significant loss of the tuberculosis inhibition indicating that an ester is required for anti-tuberculosis activity. Optimization of the isoxazoline C-5 position produced compounds with improved anti-tuberculosis activity, most notably the piperazyl-urea and piperazyl-carbamate analogs.

Pharmacokinetically-guided lead optimization of nitrofuranylamide anti-tuberculosis agents.

In an effort to develop novel and more potent therapies to treat tuberculosis, a new class of chemical agents, nitrofuranylamides, is being developed. The present study examines biopharmaceutic properties and preclinical pharmacokinetics of nitrofuranylamides at early stages of drug discovery to accelerate the optimization of leads into development candidates. The first tested compound, Lee 562, had high anti-tuberculosis activity in vitro, but exhibited poor metabolic stability resulting in a high systemic clearance, a short elimination half-life and low oral bioavailability in vivo in rats. Thus, two follow-up compounds were designed and tested that included structural modifications for increased metabolic stability. Both compounds showed improved metabolic stability compared to Lee 562, with Lee 878 being much more stable than Lee 952. As a consequence, the oral bioavailability of Lee 878 reached approximately 27% compared to 16% for the other two compounds. This observation prompted us to select compounds based on metabolic stability screening and a new set of nine compounds with high in vitro activity were tested for metabolic stability. The most stable compound in the assay, Lee 1106 was selected for further pharmacokinetic evaluation in rats. Surprisingly, Lee 1106 exhibited poor oral bioavailability, 4.6%. Biopharmaceutic evaluation of the compound showed that the compound has poor aqueous solubility and a high clogP. Based on these results, a screening paradigm was developed for optimization of the nitrofuranylamide lead compounds in a timely and cost-effective manner that might also be applicable to other classes of anti-infective drugs.

Nitrofurans as novel anti-tuberculosis agents: identification, development and evaluation.

During a search for new anti-tuberculosis agents, a screen of a commercially available library provided a hit nitrofuranyl amide. This hit was selected for further development due to its potential as an anti-tuberculosis agent with a novel mechanism of action, and its potential for activity against both actively growing and latent bacteria. This review covers the optimization of this lead and the strategies applied for developing this series into anti-tuberculosis agents. To optimize the hit, a series of libraries were synthesized, producing several compounds that showed increased anti-tuberculosis activity along with a strong structure activity relationship. The most active compounds from the first optimization series showed good in vitro anti-tuberculosis activity and limited in vivo efficacy, but their application was restricted due to solubility problems. Therefore, a second generation optimization library was designed and synthesized in order to increase bioavailability and solubility while maintaining good anti-tuberculosis activity. Hydrophilic cyclic secondary amines were substituted to the core scaffold and a benzyl piperazine substitution was found to be most effective in achieving improved solubility and potent anti-tuberculosis activity. However, bioactivity studies of these 2nd generation leads showed that the in vivo anti-tuberculosis activity of these compounds was limited due to rapid metabolism. Consequently, a 3rd generation of compounds was designed and synthesized in which potential sites of metabolism were blocked.

Synthesis of new and potent analogues of anti-tuberculosis agent 5-nitro-furan-2-carboxylic acid 4-(4-benzyl-piperazin-1-yl)-benzylamide with improved bioavailability.

Previously, the lead compound 5-nitro-furan-2-carboxylic acid 4-(4-benzyl-piperazin-1-yl)-benzylamide was identified in our anti-tuberculosis drug discovery program. Although this compound demonstrated excellent in vitro activity, it did not meet the expected in vivo profiles due to structural features that resulted in rapid metabolic cleavage and poor absorption, which therefore limited its bioavailability. In efforts to increase the bioavailability, a new series of analogues was successfully synthesized using three modification schemes: replacement of the benzyl group on the piperazine C-ring with carbamate and urea functional groups; introduction of a nitrogen atom into the aromatic ring-B; and expansion of the ring-B to a bicyclic tetrahydroisoquinoline moiety. These modifications retained strong activity and in some case gained superior anti-tuberculosis activity, increased absorption, and serum half life.