Probing binding requirements of NAD kinase with modified substrate (NAD) analogues Laurent Bonnac, a Liqiang Chen, a Rashmi Pathak, a Guangyao Gao, a Qian Ming, a Eric Bennett, a Krzysztof Felczak, a Martin Kullberg, a Steven E. Patterson, a Francesca Mazzola, b Giulio Magni b and Krzysztof W. Pankiewicz a, * a Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA b Instituto di Biotecnologie Biochimiche, Universita Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy Received 16 November 2006; revised 2 January 2007; accepted 3 January 2007 Available online 17 January 2007 Abstract—Synthesis of novel NAD + analogues that cannot be phosphorylated by NAD kinase is reported. In these analogues the C2 0 hydroxyl group of the adenosine moiety was replaced by fluorine in the ribo or arabino configuration (1 and 2, respectively) or was inverted into arabino configuration to give compound 3. Compounds 1 and 2 showed inhibition of human NAD kinase, whereas analogue 3 inhibited both the human and Mycobacterium tuberculosis NAD kinase. An uncharged benzamide adenine dinucleotide (BAD) was found to be the most potent competitive inhibitor (K i = 90 lM) of the human enzyme reported so far. Ó 2007 Elsevier Ltd. All rights reserved. NAD kinase catalyzes a magnesium-dependent phos- phorylation of the 2 0 -hydroxyl group of the adenosine ribose moiety of nicotinamide adenine dinucleotide (NAD) using ATP or inorganic polyphosphates as phos- phoryl donors to give NADP (Scheme 1). 1 There are two classes of the enzyme, one which is specific for NAD + and one which also phosphorylates NADH. The Mycobacterium tuberculosis enzyme falls into the latter category. No other pathway of NADP biosynthe- sis has been found in prokaryotic or eukaryotic cells. Although NAD kinase was discovered as early as in 1937, little was known about the structure, function, and mode of action of this enzyme until very recently. 2 NAD kinase plays a crucial role in controlling the cellu- lar redox state by regulating the ratio of reduced coen- zymes NADH/NADPH. 2,3 More recently, it has become clear that NADP-mediated signal transduction affects numerous metabolic pathways and there is a growing body of evidence that NADP serves as an important component in the control of essential cellular processes. For example, NADP is converted into nico- tinic acid adenine dinucleotide phosphate (NAADP) by the exchange of nicotinamide with nicotinic acid cat- alyzed by CD38 and ADP-ribosyl cyclase. 4–6 NAADP is the most potent Ca 2+ mobilizing agent in mammalian cells. Interestingly, human NAD-kinase is highly selec- tive and does not phosphorylate NAAD into NAADP. 7 In contrast to NAD, which serves as a substrate for a number of degradation enzymes such as ADP- ribosyl transferases, 8 poly-ADP-ribose polymerases, 9 0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2007.01.012 Keywords: NAD kinase; M. tuberculosis; Benzamide adenine dinucle- otide (BAD); NAD analogues. * Corresponding author. Tel.: +1 612 625 7968; fax: +1 612 625 8252; e-mail: panki001@umn.edu N N N N NH 2 P O HO O O OH HO O O HO OH P O OH O N CONH 2 + N N N N NH 2 P O HO O O OH O O O HO OH P O OH O N CONH 2 + P O HO HO NAD-kinase ATP or poly(P) NAD NADP Scheme 1. NAD phosphorylation by NAD-kinase. Bioorganic & Medicinal Chemistry Letters 17 (2007) 1512–1515