International Journal of Molecular Sciences Article The Biochemical Pathways of Nicotinamide-Derived Pyridones Faisal Hayat 1,2,†, Manoj Sonavane 1,2,3,†, Mikhail V. Makarov 2 , Samuel A. J. Trammell 4 , Pamela McPherson 2, Natalie R. Gassman 2,3 and Marie E. Migaud 1,2,* 1 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA; [email protected] (F.H.); [email protected] (M.S.) 2 Mitchell Cancer Institute, College of Medicine, University of South Alabama, Mobile, AL 36604, USA; [email protected] (M.V.M.); [email protected] (P.M.); [email protected] (N.R.G.) 3 Department of Physiology & Cell Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA 4 Novo Nordisk Foundation, Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark; [email protected] * Correspondence: [email protected] † Shared first authorship. Abstract: As catabolites of nicotinamide possess physiological relevance, pyridones are often in- cluded in metabolomics measurements and associated with pathological outcomes in acute kidney injury (AKI). Pyridones are oxidation products of nicotinamide, its methylated form, and its ribosy- lated form. While they are viewed as markers of over-oxidation, they are often wrongly reported or mislabeled. To address this, we provide a comprehensive characterization of these catabolites of vitamin B3, justify their nomenclature, and differentiate between the biochemical pathways that lead to their generation. Furthermore, we identify an enzymatic and a chemical process that accounts for the formation of the ribosylated form of these pyridones, known to be cytotoxic. Finally, we Citation: Hayat, F.; Sonavane, M.; demonstrate that the ribosylated form of one of the pyridones, the 4-pyridone-3-carboxamide riboside Makarov, M.V.; Trammell, S.A.J.; (4PYR), causes HepG3 cells to die by autophagy; a process that occurs at concentrations that are McPherson, P.; Gassman, N.R.; Migaud, M.E. The Biochemical comparable to physiological concentrations of this species in the plasma in AKI patients. Pathways of Nicotinamide-Derived Pyridones. Int. J. Mol. Sci. 2021, 22, Keywords: NAD; redox cofactor; nicotinamide; pyridones 1145. https://doi.org/10.3390/ijms 22031145 Academic Editor: Maurizio Battino 1. Introduction Received: 28 December 2020 Through multiple yet convergent biosynthetic pathways, all components of vitamin Accepted: 19 January 2021 B3 are precursors to the nicotinamide-derived redox cofactor, nicotinamide adenine dinu- Published: 24 January 2021 cleotide (NAD), its reduced form NADH, its phosphate parent NADP, and the reduced form NADPH, collectively referred to as NAD(P)(H) [1]. The water-soluble components Publisher’s Note: MDPI stays neutral that constitute vitamin B3 are obtained through the diet and/or supplementation. Its with regard to jurisdictional claims in regular intake must compensate for the daily losses stemming from the degradation of published maps and institutional affil- the cofactors [2]. Some of the NAD(P)(H) degradation products (Figure1) include N- iations. methyl-4-pyridone-3-carboxamide (N-Me-4PY) and N-methyl-6-pyridone-3-carboxamide, often reported as N-methyl-2-pyridone-5-carboxamide (N-Me-6PY). Both pyridones are detected in plasma and urine as major vitamin B3 degradation products and are oxidized forms of methyl-nicotinamide (Me-Nam) [3,4]. The ribosylated form of the pyridone car- Copyright: © 2021 by the authors. boxamides, 4-pyridone-3-carboxamide riboside (4PYR), and 6-pyridone-3-carboxamide Licensee MDPI, Basel, Switzerland. riboside also known as 2-pyridone-5-carboxamide riboside (6PYR), are also detected in This article is an open access article urine [5]. In blood, these ribosylated pyridones are mostly present intracellularly in distributed under the terms and their triphosphorylated (4PYR-TP and 6PYR-TP) [6,7] or adenine dinucleotidic forms [8]. conditions of the Creative Commons N-Methyl-2-pyridone-3-carboxamide (Me-2PY) and 2-pyridone-3-carboxamide riboside Attribution (CC BY) license (https:// (2PYR) are the least prominent pyridones in the literature, as they are much less often creativecommons.org/licenses/by/ detected in biological samples [9]. 4.0/). Int. J. Mol. Sci. 2021, 22, 1145. https://doi.org/10.3390/ijms22031145 https://www.mdpi.com/journal/ijms Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 1145 2 of 27 28 N-Me-Nam NAD(P)H NAD(P) 2NAD(P)O 6NAD(P)O 4NAD(P)O N-Me-2PY N-Me-6PY N-Me-4PY NQO2 NQO2 2PYR 6PYR 4PYR NR NRH 2PYR-TP 6PYR-TP 4PYR-TP Figure 1. NAD(P)(H)-derivedNAD(P)(H)-derived pyridone pyridone catabolites catabolites.. An An additional additional route route to tothe the tripho triphosphatesphate from from the the monophosphate monophosphate ri- bosylatedribosylated pyridone pyridone generated generated from from the the dinucleotides dinucleotides can can also also be envisaged. be envisaged. NAD(P)(H): NAD(P)(H): nicotinamide nicotinamide adenine adenine dinucleo- dinu- tidecleotide (phosphate)(reduced (phosphate)(reduced form); form); NAD(P)O: NAD(P)O: hyper- hyper- oxidized oxidized forms forms of NAD(P) of NAD(P) containing containing pyridone pyridone moieties; moieties; PYR-TP: PYR-TP: pyr- idonepyridone riboside riboside triphosphate; triphosphate; PYR: PYR: pyridone pyridone riboside; riboside; The The 2, 2,4, 4,and and 6 6positions positions of of the the carbonyl carbonyl unit unit are are indicated indicated in the abbreviation; N-Me-PY: N-methyl pyridone. abbreviation; N-Me-PY: N-methyl pyridone. AsAs waste products, thesethese by-productsby-products of of NAD(P)(H) NAD(P)(H) metabolism metabolism associate associate with with aging ag- ingand and nephrotic nephrotic dysfunction dysfunction as their as their levels levels associate associate with advancedwith advanced kidney kidney injuries injuries (AKI) (AKI)and kidney and kidney failure failure [4,6,10 –[4,6,10–12].12]. Liquid Liquid chromatography chromatography combined combined with mass with spectrometry mass spec- trometryis the most is commonthe most methodcommon used method to detect used nicotinamide-derivedto detect nicotinamide-derived pyridones pyridones in biological in biologicalsamples [13 samples–15]. However, [13–15]. muchHowever, confusion much arisesconfusion from arises their detection,from their identification, detection, identi- and fication,reporting and as additionalreporting as nicotinamide additional nicotina metabolitesmide that metabolites are the non-methylated that are the non-methylated or ribosylated orpyridones ribosylated are alsopyridones often mistaken are also foroften their mistak methylateden for speciestheir methylated in reports [species5,9,11,15 in– 18reports]. The [5,9,11,15–18].misperception The stems misperception from confusing stems nomenclature, from confusing compounded nomenclature, by poor compounded quantification by poordue toquantification facile fragmentation due to facile and adduct fragmentation formation and under adduct mass formation spectrometry under conditions. mass spec- trometryCritically, conditions. the chemical nature of each form of the pyridone informs on the type of catabolicCritically, pathway the thechemical nicotinamide-derived nature of each form species of the have pyridone undertaken. informs For on instance, the type only of catabolicthe methylated pathway form the of nicotinamide-derived nicotinamide (N-Me-Nam) species can have be the undertaken. precursor ofFor the instance, three methy- only thelated methylated pyridones form (N-Me-2PY, of nicotinamideN-Me-4PY, (N-Me-Nam) and N-Me-6PY, can be Figure the precursor1)[ 19–22 of]. the Similarly, three meth- only ylatedthe ribosylated pyridones form (N-Me-2PY, of nicotinamide N-Me-4PY, can beand the N-Me-6PY, source of theFigure pyridone 1) [19–22]. ribosides Similarly, [17]. Eachonly theof the ribosylated catabolic form routes of indicates nicotinamide dysregulation can be th ine source NAD-dependent of the pyridonemetabolism ribosides [23 [17].,24]. Each Pyri- ofdone the ribosides, catabolic unlikeroutes the indicates methylated dysregulation species, can in be NAD-dependent converted to the adeninemetabolism dinucleotide [23,24]. Pyridoneor the triphosphate ribosides, unlike form [the17, 25methylated]. The 4PYR species, isomer can not be onlyconverted circulates to the in adenine plasma dinu- once cleotidegenerated or butthe istriphosphate also internalized form and[17,25]. metabolized The 4PYR to isomer species not that only interfere circulates with enzymaticin plasma onceprocesses generated [26–28 but]. Possessing is also internalized a systematic, and reliablemetabolized account to species of the pyridones’ that interfere distribution with en- zymaticwould greatly processes help [26–28]. identify Possessing whether these a systemat speciesic, are reliable pathophysiologically account of the pyridones’ significant. dis- tributionHere, would we present greatly a help comprehensive identify whethe comparativer these species characterization are pathophysiologically of each species andsig- nificant.explore the mechanisms by which their under-detection and misidentification can arise. Int. J. Mol. Sci. 2021, 22, 1145 3 of 28 We also propose a mechanism for the wide-spread production of the ribosylated pyridone 4PYR and rationalize selective cytotoxicity by comparing how exogenous exposure to ribosylated pyridones affects cell survival. 2. Results The N-methylated 6-pyridone were easily generated