Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD
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biomolecules Article Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD Lars Jansen Sverkeli 1,2, Faisal Hayat 3, Marie E. Migaud 3 and Mathias Ziegler 2,* 1 Department of Biological Sciences, University of Bergen, 5007 Bergen, Norway; [email protected] 2 Department of Biomedicine, University of Bergen, 5009 Bergen, Norway 3 Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA; [email protected] (F.H.); [email protected] (M.E.M.) * Correspondence: [email protected] Abstract: It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a Citation: Sverkeli, L.J.; Hayat, F.; valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by Migaud, M.E.; Ziegler, M. Enzymatic nicotinamide phosphoribosyltransferase (NamPT). and Chemical Syntheses of Vacor + Analogs of Nicotinamide Riboside, Keywords: NAD ; vacor; base exchange; Vorbruggen chemistry NMN and NAD. Biomolecules 2021, 11, 1044. https://doi.org/10.3390/ biom11071044 1. Introduction Academic Editor: Jesús Fernandez Since its discovery in the early 1900s [1], nicotinamide adenine dinucleotide (NAD) Lucas has been considered a vital cofactor and signaling molecule for all living organisms. Orig- inally identified as a cofactor for redox reactions [1–3], it has since then been shown to Received: 1 June 2021 serve as a substrate for several important cellular functions, many of which lead to the Accepted: 7 July 2021 degradation of NAD [4–9]. As NAD is constantly consumed, the cell is dependent on a Published: 16 July 2021 continuous supply of NAD. In mammalian cells, most NAD is synthesized in a two-step process called the nicotinamide (Nam) salvage pathway (Figure1). In this process, Nam Publisher’s Note: MDPI stays neutral is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyl with regard to jurisdictional claims in published maps and institutional affil- transferase (NamPT) in a rate-limiting reaction [10]. This reaction requires phosphoribosyl iations. pyrophosphate (PRPP) as co-substrate. NMN is then converted to NAD using ATP in a reaction catalyzed by nicotinamide mononucleotide adenylyl transferases (NMNATs), of which there are three isoforms with different subcellular localization and tissue-specific expression patterns [11]. As Nam is the product of most reactions consuming NAD, the salvage pathway lets the cell recycle Nam as its main NAD precursor. Given the impor- Copyright: © 2021 by the authors. tance of NAD, the ability to control the cell’s NAD pool can be a very valuable asset Licensee MDPI, Basel, Switzerland. from a therapeutic perspective [5,12–15]. As the NAD pool is constantly replenished, one This article is an open access article distributed under the terms and promising strategy for modulating the cell’s NAD levels is interfering with NAD produc- conditions of the Creative Commons tion [15–20]. Consequently, the NAD biosynthetic pathways have emerged as a topic of Attribution (CC BY) license (https:// significant research. creativecommons.org/licenses/by/ 4.0/). Biomolecules 2021, 11, 1044. https://doi.org/10.3390/biom11071044 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1044 2 of 12 Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 13 NRKs Figure 1. VacorVacor acts acts as as an an analogue analogue to tonicotinamide: nicotinamide: Overview Overview of the of salvage the salvage pathway pathway of NAD of NADbiosynthesis biosynthesis (left). Nico- (left). tinamide is converted to nicotinamide mononucleotide (NMN) in a rate-limiting reaction catalyzed by nicotinamide phos- Nicotinamide is converted to nicotinamide mononucleotide (NMN) in a rate-limiting reaction catalyzed by nicotinamide phoribosyl transferase (NamPT). NMN is then converted to NAD in a reaction catalyzed by nicotinamide mononucleotide phosphoribosyl transferase (NamPT). NMN is then converted to NAD in a reaction catalyzed by nicotinamide mononu- adenylyl transferases (NMNATs). The alternative NAD precursor nicotinamide riboside (NR) enters the pathway by con- cleotideversion to adenylyl NMN catalyzed transferases by nicotinamide (NMNATs). The riboside alternative kinases NAD (NRKs). precursor Vacor nicotinamide is converted ribosideto an NAD (NR) analogue enters thethrough pathway the bysame conversion pathway, to being NMN converted catalyzed to by vacor nicotinamide mononucleotide riboside kinases(VMN) (NRKs).by NamPT Vacor and is then converted furthe tor converted an NAD analogue to vacor throughadenine thedinucleotide same pathway, (VAD) being by NMNAT2. converted toPossible vacor mononucleotidealternative pathways (VMN) for by generating NamPT and or thenconsuming further VAD converted that are to vacor addressed adenine in dinucleotidethe present study (VAD) are by indicated NMNAT2. with Possible question alternative marks. pathways for generating or consuming VAD that are addressed in the present study are indicated with question marks. One compound that has recently seen a certain resurgence in attention is vacor. Orig- inallyOne developed compound as a that rat haspoison recently [21],seen with aout certain a known resurgence mechanism in attention of action, is vacor. recent Origi- re- searchnally developed has shown as that a rat vacor poison supplementation [21], without a knownleads to mechanism accumulation of action, of vacor recent mononucle- research otidehas shown (VMN) that and vacor vacor supplementation adenine dinucleotide leads (VAD), to accumulation that is, vacor-containing of vacor mononucleotide analogues of(VMN) NMN and and vacor NAD, adenine respectively dinucleotide [20,22]. (VAD),This conversion that is, vacor-containing is catalyzed by analoguesNamPT and of NMNAT2NMN and [20,22], NAD, respectivelyshowing that [20 vacor,22]. indeed This conversion enters the issalvage catalyzed pathway by NamPT of the andNAD NM- bi- osynthesisNAT2 [20,22 (Figure], showing 1). Interestingly, that vacor indeed vacor enters has been the salvage shown pathwayto act as ofa thepotent NAD cytotoxic biosyn- agent,thesis (Figureleading1 ).to Interestingly,rapid NAD depletion vacor has and been subsequent shown to cell act asdeath a potent [20,22]. cytotoxic Furthermore, agent, vacor-inducedleading to rapid cytotoxicity NAD depletion appears and to subsequentbe selective, cellaffecting death primarily [20,22]. Furthermore, neuronal and vacor- pan- creaticinduced tissue cytotoxicity [20,22,23], appears making to bevacor selective, an interesting affecting primarilytoxin not neuronalonly from and an pancreaticacademic viewpoint,tissue [20,22 but,23 ],also making as a potential vacor an chemotherapeutic interesting toxin not agent. only The from molecular an academic mechanisms viewpoint, of vacor-inducedbut also as a potential cytotoxicity chemotherapeutic have remained agent.unknown. The However, molecular it mechanismsis clear that the of vacor-accu- induced cytotoxicity have remained unknown. However, it is clear that the accumulation mulation of vacor-derived NAD analogs are crucial elements [20,22]. Understanding these of vacor-derived NAD analogs are crucial elements [20,22]. Understanding these mecha- mechanisms could identify novel biological processes and eventually new therapeutic tar- nisms could identify novel biological processes and eventually new therapeutic targets. gets. Critically, current methods to produce vacor analogs of NAD and its biosynthetic Critically, current methods to produce vacor analogs of NAD and its biosynthetic interme- intermediates are rather complex and time consuming, as they involve expression and diates are rather complex and time consuming, as they involve expression and purification purification of the enzymes of the salvage pathway of the NAD biosynthesis [20,22]. In of the enzymes of the salvage pathway of the NAD biosynthesis [20,22]. In this study, this study, we developed an easy, one-step method for synthesizing VAD (Figure 1), ex- we developed an easy, one-step method for synthesizing VAD (Figure1), exploiting the ploiting the catalytic properties of commercially available ADP-ribosyl cyclases [24,25]. catalytic properties of commercially available ADP-ribosyl cyclases [24,25]. Biomolecules 2021, 11, 1044 3 of 12 In addition, while the salvage pathway is the main source of NAD for the cell, alterna- tive NAD precursors can be converted to NAD through different pathways. Nicotinamide riboside (NR), in particular, has been extensively studied recently due to its ability to boost systemic NAD levels