Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues Riccardo Petrelli a , Yuk Yin Sham a , Liqiang Chen a , Krzysztof Felczak a , Eric Bennett a , Daniel Wilson a , Courtney Aldrich a , Jose S. Yu a , Loredana Cappellacci b , Palmarisa Franchetti b , Mario Grifantini b , Francesca Mazzola c , Michele Di Stefano c , Giulio Magni c , Krzysztof W. Pankiewicz a, * a Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA b Dipartimento di Scienze Chimiche, Universita’ di Camerino, 62032 Camerino, Italy c Instituto di Biotecnologie Biochimiche, Universita’ Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy article info Article history: Received 6 May 2009 Revised 3 June 2009 Accepted 6 June 2009 Available online 13 June 2009 Keywords: NAD kinase NADP Enzyme inhibition Tiazofurin Dithioadenosine analogues, NAD conformation abstract Diadenosine disulfide (5) was reported to inhibit NAD kinase from Lysteria monocytogenes and the crystal structure of the enzyme–inhibitor complex has been solved. We have synthesized tiazofurin adenosine disulfide (4) and the disulfide 5, and found that these compounds were moderate inhibitors of human NAD kinase (IC 50 = 110 lM and IC 50 = 87 lM, respectively) and Mycobacterium tuberculosis NAD kinase (IC 50 = 80 lM and IC 50 = 45 lM, respectively). We also found that NAD mimics with a short disulfide (–S–S–) moiety were able to bind in the folded (compact) conformation but not in the common extended conformation, which requires the presence of a longer pyrophosphate (–O–P–O–P–O–) linkage. Since majority of NAD-dependent enzymes bind NAD in the extended conformation, selective inhibition of NAD kinases by disulfide analogues has been observed. Introduction of bromine at the C8 of the adenine ring restricted the adenosine moiety of diadenosine disulfides to the syn conformation making it even more compact. The 8-bromoadenosine adenosine disulfide (14) and its di(8-bromoadenosine) analogue (15) were found to be the most potent inhibitors of human (IC 50 =6 lM) and mycobacterium NAD kinase (IC 50 = 14–19 lV reported so far. None of the disulfide analogues showed inhibition of lactate-, and ino- sine monophosphate-dehydrogenase (IMPDH), enzymes that bind NAD in the extended conformation. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Until recently, between the two pyridine nucleotides, nicotin- amide adenine dinucleotide (NAD) has received much more atten- tion than its phosphorylated counterpart, nicotinamide adenine dinucleotide 2 0 -phosphate (NADP). 1,2 NAD plays a crucial role in cellular catabolic conversions, 3,4 whereas the major role of NADPH is to control the oxidative state of the cell and stand guard against the ‘oxidative stress’. 5,6 NAD kinase is a key enzyme that regulates supply of NADP in the cell. 7 Human NAD kinase catalyzes a magnesium-dependent phosphorylation of the 2 0 -hydroxyl group of the adenosine ribose moiety of NAD using adenosine triphosphate (ATP) as phosphoryl donor to produce NADP (Scheme 1). Thus, in contrast to the family of ATP-dependent protein kinases that phosphorylate serine, thre- onine, or tyrosine residues, NAD kinases bind both ATP and NAD. Majority of protein kinase inhibitors have been designed to bind at the ATP-binding domain, 8 however, this domain is not a good target for the design of potential NAD kinase inhibitors. It is solvent exposed and does not interact with the ATP closely. Chelation with Mg 2+ is needed to engage ATP at the catalytic domain. In contrast, the NAD binding domain, as a substrate binding site, is ideal as it plays a crucial role in molecular recognition of NAD and its analogues. Bacterial enzymes can use inorganic polyphosphates as phos- phoryl donors (Scheme 1) in addition to ATP. An extensive biochem- ical, enzymatic, and structural characterization of Mycobacterium tuberculosis NAD kinase have been recently reported. 9–11 This kinase is of special interest as a new target for the development of potential drugs against multi-drug resistant (MDR) tuberculosis (TB). The mycobacterium enzyme requires a millimolar concentration of NAD (K m = 3.3 mM), which is sixfold higher than that of human en- zyme (0.5 mM). Thus, low micromolar inhibitors would be sufficiently competitive to suppress NAD kinase activity, especially that of the M. tuberculosis enzyme. Inhibitors of the human enzyme are also of interest. They should indirectly reduce critical supply of NADPH. 1,6 Although NAD kinase provides mainly oxidized form NADP + (phosphoryla- tion of NADH by the human enzyme is much less efficient), this 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.06.013 * Corresponding author. Tel.: +1 612 625 7968; fax: +1 612 624 825. E-mail address: panki001@umn.edu (K.W. Pankiewicz). Bioorganic & Medicinal Chemistry 17 (2009) 5656–5664 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc