A fluorescent assay suitable for inhibitor screening and vanin tissue quantification

Ruan, Benfang; Cole, Derek C.; Wu, Paul W.; Quazi, Amira; Page, Karen; Wright, Jill F.; Huang, Nelson; Stock, Joseph R.; Nocka, Karl; Aulabaugh, Ann; Krykbaev, Rustem; Fitz, Lori; Wolfman, Neil M.; Fleming, Margaret

doi:10.1016/j.ab.2009.12.010

Cited by 40 publications

(54 citation statements)

References 20 publications

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“…We used pantothenate-AMC as substrate at 37°C for 30 min to assay the pantetheinase activity; the hydrolysis catalyzed by VNN1 yields pantothenic acid and detectable free fluorescent AMC (excitation 340 nm; emission 460 nm) (24). The liver extracts were washed three times with PBS and lysed with potassium phosphate buffer (100 mmol/L, pH 7.5) containing 0.1% Triton X-100 and 0.6% sulfosalicylic acid.…”

Section: Pantetheinase Activity Assaymentioning

confidence: 99%

Vanin-1 Is a Key Activator for Hepatic Gluconeogenesis

et al. 2014

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Vanin-1 (VNN1) is a liver-enriched oxidative stress sensor that has been implicated in the regulation of multiple metabolic pathways. Clinical investigations indicated that the levels of VNN1 were increased in the urine and blood of diabetic patients, but the physiological significance of this phenomenon remains unknown.In this study, we demonstrated that the hepatic expression of VNN1 was induced in fasted mice or mice with insulin resistance. Gain-and loss-of-function studies indicated that VNN1 increased the expression of gluconeogenic genes and hepatic glucose output, which led to hyperglycemia. These effects of VNN1 on gluconeogenesis were mediated by the regulation of the Akt signaling pathway. Mechanistically, vnn1 transcription was activated by the synergistic interaction of peroxisome proliferator-activated receptor g coactivator 1a (PGC-1a) and hepatocyte nuclear factor-4a (HNF-4a). A chromatin immunoprecipitation analysis indicated that PGC-1a was present near the HNF-4a binding site on the proximal vnn1 promoter and activated the chromatin structure. Taken together, our results suggest an important role for VNN1 in regulating hepatic gluconeogenesis. Therefore, VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis.

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Section: Pantetheinase Activity Assaymentioning

confidence: 99%

Vanin-1 Is a Key Activator for Hepatic Gluconeogenesis

et al. 2014

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“…In a previous study, we showed that vanins are ubiquitously expressed (16). Using an aminomethylcoumarine derivative of pantothenic acid, which is a pantothenamide with a fluorescent leaving group as a substrate (19), pantetheinase activity in serum is readily detected. We found that this hydrolytic activity was completely abolished by heat inactivation of the serum (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A recent publication identified pantetheinase inhibitors derived from various chemical scaffolds in the LOPAC library, via a high-throughput approach (19). These compounds are all nonselective and have modest potency.…”

Section: Discussionmentioning

confidence: 99%

“…Pantetheinase activity was measured by the amount of free 7-amino-4-methylcoumarin (AMC) released by the hydrolysis of the pantetheine analogue pantothenate AMC (19). Pantothenate AMC (final concentration, 10 M) was incubated in phosphate buffer (100 mM potassium phosphate buffer pH 8.0) in the presence of serum (1%) as the pantetheinase source with or without a potential pantetheinase inhibitor.…”

Section: Bacterial Growth Assays Strains Of Escherichia Coli Staphymentioning

confidence: 99%

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Combination of Pantothenamides with Vanin Inhibitors as a Novel Antibiotic Strategy against Gram-Positive Bacteria

Jansen

Hermkens

Zeeuwen

et al. 2013

Antimicrob Agents Chemother

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eThe emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Grampositive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents. There is a continuous battle between humans and microorganisms, and the development of resistance against current antibiotics creates a need to develop new drug scaffolds and targets (1). There are several classes of antibiotic-resistant pathogens that are emerging as major threats. Methicillin-resistant and vancomycin-resistant Staphylococcus aureus strains (MRSA and VRSA) and multidrug-resistant Streptococcus pneumoniae are serious health problems in both hospital settings and the community. Multidrug-resistant Gram-negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) are a second group of organisms that pose a threat of untreatable infections. Finally, poverty-related conditions such as tuberculosis and parasitic diseases, which are particularly important in developing countries, are a cause of great concern, as the already-limited number of treatment modalities is decreasing because of resistance. The development of new antibiotics has been slow, and most compounds to date have been derived from a limited number of molecular scaffolds targeting a few microbial molecular pathways. Discovery of new scaffolds and new targets should clearly be a priority, although the commercial prospects of antibiotic development are unfavorable (1).In 1970, amides derived from pantothenic acid (vitamin B5), were reported to possess antibiotic activity in vitro (2). During the last few decades, many of these pantothenamides have been synthesized and the pu...

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“…Pantetheinases, also referred to as vanins [44], are enzymes that hydrolyse pantetheine into pantothenate and cysteamine. These enzymes had been shown previously to accept amide analogues of pantothenate other than pantetheine as substrates for hydrolysis [45][46][47] (Figure 3). The presence of pantetheinase activity in the parasite culture medium (as a result of the addition of Albumax II, a serum substitute, or human serum) resulted in pantothenamide degradation and abrogation of the potent antiplasmodial effect [41].…”

Section: Pantothenamidesmentioning

confidence: 98%

Exploiting the coenzyme A biosynthesis pathway for the identification of new antimalarial agents: the case for pantothenamides

Saliba

Spry

2014

Biochemical Society Transactions

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Malaria kills more than half a million people each year. There is no vaccine, and recent reports suggest that resistance is developing to the antimalarial regimes currently recommended by the World Health Organization. New drugs are therefore needed to ensure malaria treatment options continue to be available. The intra-erythrocytic stage of the malaria parasite's life cycle is dependent on an extracellular supply of pantothenate (vitamin B5), the precursor of CoA (coenzyme A). It has been known for many years that proliferation of the parasite during this stage of its life cycle can be inhibited with pantothenate analogues. We have shown recently that pantothenamides, a class of pantothenate analogues with antibacterial activity, inhibit parasite proliferation at submicromolar concentrations and do so competitively with pantothenate. These compounds, however, are degraded, and therefore rendered inactive, by the enzyme pantetheinase (vanin), which is present in serum. In the present mini-review, we discuss the two strategies that have been put forward to overcome pantetheinase-mediated degradation of pantothenamides. The strategies effectively provide an opportunity for pantothenamides to be tested in vivo. We also put forward our 'blueprint' for the further development of pantothenamides (and other pantothenate analogues) as potential antimalarials.

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A fluorescent assay suitable for inhibitor screening and vanin tissue quantification

Cited by 40 publications

References 20 publications

Vanin-1 Is a Key Activator for Hepatic Gluconeogenesis

Vanin-1 Is a Key Activator for Hepatic Gluconeogenesis

Combination of Pantothenamides with Vanin Inhibitors as a Novel Antibiotic Strategy against Gram-Positive Bacteria

Exploiting the coenzyme A biosynthesis pathway for the identification of new antimalarial agents: the case for pantothenamides

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