A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

Sharma, Lalit Kumar; Leonardi, Roberta; Lin, Wenwei; Boyd, Vincent A.; Goktug, Asli N.; Shelat, Anang A.; Chen, Taosheng; Jackowski, Suzanne; Rock, Charles O.

doi:10.1021/jm501585q

Cited by 28 publications

(24 citation statements)

References 20 publications

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“…Acyl-carnitines are an established activating ligand (8), and other signaling molecules like the N-acylethanolamines (7) may also play a role. We have previously demonstrated that small molecule effectors can modulate the activity of PANK3 in a manner that appears to be allosteric in nature (7,30). The studies reported here have confirmed the allosteric nature of PANK3 and now provide the framework for a structurebased optimization of these small molecule modulators of pantothenate kinase with potential therapeutic applications.…”

Section: Discussionsupporting

confidence: 69%

Allosteric Regulation of Mammalian Pantothenate Kinase

Subramanian

Yun²,

Yao

et al. 2016

Journal of Biological Chemistry

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Pantothenate kinase catalyzes the key regulatory step in CoA biosynthesis in bacteria and mammals (1-3). In mammals, there are four distinct kinases that exhibit tissue-specific expression as follows: PANK1␣ and PANK1␤ are splice variants of the PANK1 gene, and PANK2 and PANK3 are encoded by the PANK2 and PANK3 genes, respectively (4, 5). The kinase isoforms possess almost identical catalytic cores but differ in their amino termini that direct the enzymes to different subcellular compartments (6). Isoform 1␣ is targeted to the nucleus, whereas isoform 1␤ is associated with endosomes in humans and mice. Isoform 2 of mice is cytosolic, but the PANK2 gene in humans encodes a protein with both nuclear localization and mitochondrial targeting sequences. Isoform 3 is cytosolic in both species. All pantothenate kinases operate by a compulsory ordered mechanism with ATP⅐Mg 2ϩ as the leading substrate followed by pantothenate ( Fig. 1) (7). The principal mechanism for controlling mammalian pantothenate kinase activity is through feedback inhibition by acetyl-CoA, and the isoforms differ in their sensitivity to this regulator ( Fig. 1) (4, 8, 9). The 1␣ and 1␤ isoforms are least sensitive to inhibition, whereas isoforms 2 and 3 are more potently inhibited by acetyl-CoA. Acetyl-CoA inhibition is competitive with ATP⅐Mg 2ϩ , but acetyl-CoA binds far more tightly than ATP (10). Acyl-carnitines antagonize the inhibition of pantothenate kinases by acetyl-CoA (8).The importance of pantothenate kinase to mammalian physiology is highlighted by the phenotypes of knock-out mice and their connection to human disease. Isoform 1 is most highly expressed in the liver, and Pank1 Ϫ/Ϫ mice have lower total hepatic CoA and exhibit fatty acid ␤-oxidation and glucose homeostasis defects in the fasted state (11). In addition, Pank1 Ϫ/Ϫ Lep Ϫ/Ϫ mice have dramatically lower blood glucose and insulin levels compared with their diabetic Lep Ϫ/Ϫ counterparts, which highlights the connection between isoform 1 and glucose homeostasis (12). Consistent with this, an association between polymorphisms in the PANK1 gene and insulin levels was uncovered in a cohort of individuals in Finland (13). It has also been shown that the severe neurodegenerative disease pantothenate kinase-associated neurodegeneration arises from mutations in the human PANK2 gene (14). Unfortunately, Pank2 Ϫ/Ϫ mice do not recapitulate the pantothenate kinaseassociated neurodegeneration disease phenotype (15,16), and this may be due either to differences in the levels of isoform expression in mouse and human brains or to the fact that human PANK2 accumulates in the intra-membrane space in mitochondria, whereas mouse isoform 2 is cytosolic (9). Finally, Pank1 Ϫ/Ϫ Pank2 Ϫ/Ϫ double knock-out mice are unable to metabolize fats and ketones resulting in early postnatal death (16), and Pank1 Ϫ/Ϫ Pank3 Ϫ/Ϫ and Pank2 Ϫ/Ϫ Pank3 Ϫ/Ϫ double knock-out mice are both embryonic lethal. A chemical knockout of all pantothenate kinases in adult mice resulted in an 80% reduction in hepatic CoA levels a...

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Section: Discussionsupporting

confidence: 69%

Allosteric Regulation of Mammalian Pantothenate Kinase

Subramanian

Yun²,

Yao

et al. 2016

Journal of Biological Chemistry

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“…Moreover, the presence/absence of mitochondrial mutations, the activity of PanK isoforms, and their localization in different cell compartments and organs are also expected to contribute to the differences in tissue concentrations of CoA and acetyl-CoA. As noted in the Introduction, PanK is an important factor in tissue-selective CoA distribution [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 36 , 37 , 38 ].…”

Section: Discussionmentioning

confidence: 99%

“…For example, the biosynthesis of CoA is controlled in part by the activity of the isoenzymes (isoforms) pantothenate kinase 1 and pantothenate kinase 2 (PanK1 and PanK2). Mutations of the PanK1 and PanK2 genes are associated with PanK-dependent neurodegeneration (PKAN) and diabetes [ 1 , 9 ]. It has been suggested that the development of modulators of PanK activity represent a promising approach to the treatment of both PKAN and diabetes [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection

et al. 2017

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Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation <1% for standard solutions, and 1–3% for deproteinized biological samples. Recovery is 95–97% for liver extracts spiked with Co-A and acetyl-CoA. Many factors may influence the tissue concentrations of CoA and acetyl-CoA (e.g., age, fed, or fasted state). Nevertheless, the values obtained by the present HPLC method for the concentration of CoA and acetyl-CoA in selected rodent tissues are in reasonable agreement with literature values. The concentrations of CoA and acetyl-CoA were found to be very low in rat plasma, but easily measurable by the present HPLC method. The method should be useful for studying cellular energy metabolism under normal and pathological conditions, and during targeted drug therapy treatment.

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“…Here, we report the development of a drug capable of allosterically activating the alternate PANK isoforms as a potential PKAN therapeutic. The lead pantazine, PZ-2891, arose from the LipE-guided chemical optimization of a hit from a high-throughput screen designed to identify inhibitors and activators of PANK3 33 , 34 . Due to the high cooperativity of the PANK dimer 9 the binding of PZ-2891 to one protomer locks the opposite protomer in a constitutively active state that is refractory to feedback inhibition by acyl-CoA.…”

Section: Introductionmentioning

confidence: 99%

A therapeutic approach to pantothenate kinase associated neurodegeneration

et al. 2018

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Pantothenate kinase (PANK) is a metabolic enzyme that regulates cellular coenzyme A (CoA) levels. There are three human PANK genes, and inactivating mutations in PANK2 lead to pantothenate kinase associated neurodegeneration (PKAN). Here we performed a library screen followed by chemical optimization to produce PZ-2891, an allosteric PANK activator that crosses the blood brain barrier. PZ-2891 occupies the pantothenate pocket and engages the dimer interface to form a PANK•ATP•Mg2+•PZ-2891 complex. The binding of PZ-2891 to one protomer locks the opposite protomer in a catalytically active conformation that is refractory to acetyl-CoA inhibition. Oral administration of PZ-2891 increases CoA levels in mouse liver and brain. A knockout mouse model of brain CoA deficiency exhibited weight loss, severe locomotor impairment and early death. Knockout mice on PZ-2891 therapy gain weight, and have improved locomotor activity and life span establishing pantazines as novel therapeutics for the treatment of PKAN.

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A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

Cited by 28 publications

References 20 publications

Allosteric Regulation of Mammalian Pantothenate Kinase

Allosteric Regulation of Mammalian Pantothenate Kinase

Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection

A therapeutic approach to pantothenate kinase associated neurodegeneration

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