Vol. 50 No. 4/2003

947–972

QUARTERLY

Review

. Analogs of diadenosine tetraphosphate (Ap4A)

Andrzej Guranowski½

Department of Biochemistry and Biotechnology, University of Agriculture, Poznañ, Poland Received: 03 September, 2003; revised: 26 November, 2003; accepted: 04 December, 2003

Key words: diadenosine tetraphosphate, Ap4A, dinucleoside polyphosphates, Ap4A analogs, biological effects of Ap4A analogs

This review summarizes our knowledge of analogs and derivatives of diadenosine 1 4 5¢,5¢¢¢-P ,P -tetraphosphate (Ap4A), the most extensively studied member of the 1 n dinucleoside 5¢,5¢¢¢-P ,P -polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4N¢sin the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N¢). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.

Dinucleoside 5¢,5¢¢¢-P1,Pn-polyphosphates cally during cellular stress, reaching sub- (NpnN¢s; where N and N¢ are nucleosides and millimolar in some cases (Lee et al., 1983; n represents the number of phosphate resi- Coste et al., 1987; Pálfi et al., 1991). Diade- dues in the polyphosphate chain that links N nosine tri- and tetraphosphates appear to be with N¢) have been found in various organ- the most prominent but diadenosine penta- isms (Garrison & Barnes, 1992). Their nor- (Pintor et al., 1992a), hexa- (Pintor et al., mal, submicromolar levels increase dramati- 1992b) as well as di- (Luo et al., 1999) and

.This work was supported by grant PBZ-KBN 059/T09/04 from the State Committee for Scientific Re- search (KBN, Poland) ½Corresponding address: Andrzej Guranowski, Katedra Biochemii i Biotechnologii, Akademia Rolnicza, ul. Wo³yñska 35, 60-637 Poznañ, Poland; phone: (48 61) 848 7201; fax: (48 61) 848 7146; e-mail: [email protected] 948 A. Guranowski 2003 heptaphosphates (Jankowski et al., 1999) and Ap5U, by transfer of the UMP residue have been detected in some mammalian cells. from UDP-glucose on to ATP and p4A, respec- In vitro studies suggest that the following en- tively. (Guranowski et al., 2004). zymes from various phyla may be responsible The biological roles of NpnN¢s are rather ob- for the accumulation of the adenine-contain- scure. Some data suggest that they act as sig- ing NpnN¢s in vivo: certain aminoacyl-tRNA nalling molecules in, for example, regulation synthetases (EC 6.1.1.x) (Zamecnik et al., of the cell cycle (Grummt, 1978; Nishimura, 1966), Ap4A phosphorylase (EC 2.7.7.53) 1998). Under certain circumstances, e.g. (Guranowski et al., 1988), firefly luciferase when competing with ATP in ATP-dependent (EC 1.13.12.7) (Guranowski et al., 1990), reactions (Rotllan & Miras-Portugal, 1985; acyl-CoA synthetase (EC 6.2.1.8) (Fontes et Pype & Slegers, 1993) and/or binding with al., 1998), HIV-1 reverse transcriptase (EC nucleotide receptors (Pintor et al., 1991), 2.7.7.49) (Meyer et al., 1998), DNA ligases NpnN¢s can be detrimental to the organism (EC 6.5.1.1) (Madrid et al., 1998; McLennan, (McLennan, 2000). The levels of NpnN¢s can 2000; Günther Sillero et al., 2002), RNA be precisely regulated by numerous degra- ligase (EC 6.5.1.3) (Atencia et al., 1999), dative enzymes (Guranowski, 2000). In addi- nonribosomal peptide synthetase (Dieckman tion to the non-specific ones, like nucleotide et al., 2001) and, in plants, coumarate-CoA pyrophosphatases/phosphodiesterases (Jaku- synthetase (EC 6.2.1.12) (Pietrowska-Borek et bowski & Guranowski, 1983; Bartkiewicz et al., 2003). A GTP:GTP guanylyltransferase al., 1984; Cameselle et al., 1984; Gasmi et al., (EC 2.7.7.45) also exists in certain organisms 1998; Vollmayer et al., 2003), for which and may be responsible for the synthesis of NpnN¢s are very good substrates, there are GpnNs, i.e. -containing NpnN¢s various specific enzymes. In higher euka- (Wang & Shatkin, 1984; Liu & McLennan, ryotes (animals and plants) there is a di- 1994). These enzymes are particularly effec- nucleoside triphosphatase (EC 3.6.1.29) that tive in the synthesis of dinucleoside preferentially converts Np3N¢s to nucleoside tetraphosphates: diadenosine tetraphosphate mono- and diphosphates, NMP + N¢DP and/or (AppppA or Ap4A) and diguanosine tetra- N¢MP + NDP, and a dinucleoside tetraphos- phosphate (GppppG or Gp4G), respectively. phatase (EC 3.6.1.17) that asymmetrically They are also able to synthesize NpnN¢sin hydrolyzes Np4N¢s to either NTP + N¢MP or which n>4. Not all of them, however, can pro- N¢TP + NMP. In lower eukaryotes — fungi duce Np3N¢s. For example, firefly luciferase (yeast) and protozoa (Euglena) — dinucleoside and coumarate-CoA synthetase are incapable tetraphosphates are degraded phosphoroly- of Ap3A synthesis. Finally, it has been sug- tically, either to N¢DP + NTP or to NDP + gested (Guranowski et al., 1990) that N¢TP, by Ap4A phosphorylases (EC 2.7.7.53). dinucleotides such as Ap4U and Ap4C, found In , Np4N's are hydrolyzed symmetri- 2+ among the -containing Np4N¢sin cally to NDP + N¢DP by a specific Co -de- Escherichia coli and Saccharomyces cerevisiae pendent Ap4A hydrolase (EC 3.6.1.41). Re- (Coste et al., 1987), can be formed by enzy- cently, however, an asymmetrically-acting matic transfer of UMP- and CMP-moieties Np4N-ase related to the higher eukaryotic en- onto ATP from appropriate intermediates in- zyme has been detected in several bacteria volved in carbohydrate and lipid metabolism (Conyers & Bessman, 1999; Cartwright et al., (e.g. UDP-glucose and CDP-choline, respec- 1999; Bessman et al., 2001; Lundin et al., tively). In fact, the triphosphate:glu- 2003). The asymmetrical Np4N-ases belong to cose-1-phosphate uridylyltransferase (EC the “nudix” protein family, comprising en- 2.7.7.9) from S. cerevisiae has been recently zymes that hydrolyze nucleotides in which a found to synthesize UpnNs, including Ap4U nucleoside diphosphate is attached to one of Vol. 50 Ap4A analogs 949 various groups assigned as x (Bessman et al., sugar moieties. NpnN¢s, including Ap4A, 1996; 2001). These nudix proteins have a con- were observed by John Moffatt and co-work- served amino+acid sequence that directly par- ers in 1965 as highly stable by-products of ticipates in catalysis (Harris et al., 2000; ATP dismutation reactions (Reiss & Moffatt, Maksel et al., 2001). A search for nudix pro- 1965). Subsequently, procedures directed to- teins in various genomes has led to the discov- wards the syntheses of various homo- (N = N¢) ery of hydrolases that prefer Ap5A and/or and hetero- (N N¢)NpnN¢s were developed Ap6A as substrates in budding yeast (S. ce- in several laboratories (Feldhaus et al., 1975; revisiae) (Cartwright & McLennan, 1999), fis- Tarussova et al., 1986; Ng & Orgel, 1987; sion yeast (Schizosaccharomyces pombe) Fukuoka et al., 1995). Other laboratories fo- (Ingram et al., 1999) and (Safrany et cused on syntheses that yielded analogs modi- al., 1999). One approach to understanding fied in the polyphosphate chain. These com- 2 3 1 2 the biological roles of NpnN¢s is through the prised: (i) replacement of the P -P (2), P -P use of structural analogs in biochemical and (3)orP1-P2 and P3,P4 (4) bridging oxygen(s) physiological studies. This review focuses on with methylene (2–4), halomethylene- (5), analogs of diadenosine 5¢,5¢¢¢-P1,P4-tetraphos- ethylene- or acetylene- group(s) (Tarussova et phate (1)* (Ap4A), the most widely investi- al., 1983; 1985; Blackburn et al., 1987a); (ii) gated member of the NpnN¢s, and presents replacement of the oxygen(s) with imido our current knowledge of both chemically and group(s) (Shumiyanzeva & Poletaev, 1984); enzymatically produced nucleotides. Particu- (iii) attachment of adenylate moieties to lar attention is paid to compounds that have methanetrisphosphonate (10) (Liu et al., been useful in studies of enzymes involved in 1999); (iv) adenylylation of polyalcohols such the metabolism of Ap4A. Analogs that behave as glycerol, erythritol (11) and pentaery- either as alternative substrates of Ap4A-de- thritol (Baraniak et al., 1999) and (v) adeny- grading enzymes and/or as enzyme inhibitors lylation of the hydroxymethyl groups of have helped to establish the mechanism of ac- di(hydroxymethyl)phosphinic acid (Baraniak tion of these enzymes. They have also in- et al., 1999). Yet another group of chemically creased the sensitivity of some enzyme as- generated Ap4A analogs comprised mono- says, or, by forming stable enzyme:ligand (6–7) (Haikal et al., 1989; Puri et al., 1995) complexes, allowed an analysis of the sub- and diphosphorothioates either of Ap4A(8) strate-binding site in the three-dimensional (Blackburn et al., 1987b), or of the aforemen- structures of certain hydrolases. This work tioned Ap4A analogs with methylene or partially updates two earlier reviews (Black- halomethylene group(s) (9) (Blackburn & burn et al., 1992; Guranowski, 2000) that pre- Guo, 1990), or of the derivatives of polyols sented some chemical and biological aspects (12–14) (Baraniak et al., 1999) and di(hydro- of both Ap4A and Ap3A analogs. xymethyl)phosphinic acid (Baraniak et al., 1999; Walkowiak et al., 2002). Recently, di(-5'-O-phosphorodithioate),di(hy- CHEMICALLY SYNTHESIZED droxymethyl)phosphonic acid (15) was syn- ANALOGS OF Ap4A thesized (Walkowiak et al., 2002). The list of Ap4A analogs can be further ex- Organic chemists have produced a wide vari- tended by modifications of the adenine or ety of Ap4A analogs differing from Ap4Aei- sugar moieties. Treatment of Ap4A with ther in the oligophosphate chain (thus being 2-chloroacetaldehyde yields the fluorescent 6 Ap4A homologs), in the base, and/or in the mono- and di(1, N -etheno)derivatives, eAp4A

*Numbers in bold refer to compounds whose structures are presented in Table 1. 950 A. Guranowski 2003

Table 1. Selected analogs of diadenosine tetraphosphate Vol. 50 Ap4A analogs 951

Table 1. Continued 952 A. Guranowski 2003

Table 1. Continued Vol. 50 Ap4A analogs 953

Table 1. Continued 954 A. Guranowski 2003

Table 1. Continued Vol. 50 Ap4A analogs 955

Table 1. Continued 956 A. Guranowski 2003

Table 1. Continued

and eAp4eA(17) (Wierzchowski et al., 1985; 3¢-hydroxyl of the (s) via an ether link- Rotllán et al., 1991). Periodate converts Ap4A age: mAp5A and mAp5mA(26) (Reinstein et into its (bis)2¢,3¢-dialdehyde derivative, al., 1990) and mAp5T (Lavie et al., 1998). oAp4oA (25), which can serve as an affinity label (Grummt et al., 1979). A chromogenic analog, in which 5-bromo-4-chloro-3-indolyl ENZYMATICALLY PRODUCED Ap4A phosphate is coupled to ATP (19) (Garrison et ANALOGS al., 1993), another fluorescent analog, lin-benzoAp4A(18) (VanDerLijn et al., 1979), Enzymatically produced Ap4A analogs can and two analogs for affinity labeling, be synthesized as a result of the broad sub- 8-azido-Ap4A(20) (Prescott & McLennan, strate specificity of some ligases, transferases 1990; Baxi et al., 1994) and pyridoxal-p4A and firefly luciferase. This ability is con- (21) (Yagami et al., 1988), have also been syn- ferred principally by the adenylate-binding thesized. sites of these enzymes. Certain amino- So far, only three Ap4A analogs modified in acyl-tRNA synthetases (Randerath et al., the sugar (ribose) moieties have been synthe- 1966; Plateau & Blanquet, 1982; Jakubowski, sized. These are the dinucleotides containing 1983; Theoclitou et al., 1994), yeast Ap4A fluorescent N-methylanthraniloyl group(s) phosphorylase (Brevet et al., 1987; Gura- (abbreviated here as m) bound to the 2¢-or nowski et al., 1988; £a¿ewska & Guranowski, Vol. 50 Ap4A analogs 957

1990), firefly luciferase (Günther Sillero et removal of a dideoxynucleotide residue from al., 1991; Ortiz et al., 1993; Günther Sillero et the 3¢-end of a chain-terminated DNA primer al., 1997) and coumarate:CoA ligase (Piet- through production of a dinucleoside poly- rowska-Borek et al., 2003) can therefore be phosphate. employed for the synthesis of different Ap4Ns Other Ap4A derivatives modified on the using ATP (or ADP in the case of the Ap4A sugar moiety can be obtained from reactions phosphorylase) as an adenylate donor and supported by 2¢,5¢-poly(A) synthase, which in- various NTPs as adenylate acceptors. In addi- troduces (deoxy)adenylate residue(s) onto the tion to NTPs, ADP can also act as an 2¢-(and 2¢¢¢-) position(s) of Ap4A(27) (Cayley adenylate acceptor (but only in the case of & Kerr, 1982; Guranowski et al., 2000) while aminoacyl-tRNA ligases) to yield Ap3A, while the recently reported poly(A) polymerase p4Aorp4G give rise to Ap5A and Ap5G, re- from E. coli can monoadenylate Ap4A at one spectively. ATPaS used as an adenylate ac- of its two 3¢-positions (28)(Günther Sillero et ceptor yields a monophosphorothioate deriva- al., 2001; Guranowski et al., 2003). tive of Ap4A (Ap4AaSorApspppA) (6,7) and The adenine-containing analogs can be suc- b,g[CH2]ATP and a,b[CH2]ATP allow the syn- cessively converted into their hypoxanthi- thesis of AppCH2ppA (2) and ApCH2pppA ne-containing counterparts, IpnNs, such as (3), respectively. eATP used as the only NTP Ip4A(16) and Ip4I by treatment with adeno- in the reaction mixture yields eAp4eA(17), sine-phosphate deaminase (EC 3.5.4.17) from while ATPgS, being a poor adenylate accep- the snail Helix pomatia or the fungus tor, yields only small amounts of Ap4AbS Aspergillus oryzae (Guranowski et al., 1995). (AppsppA) (Günther Sillero et al., 1991). Finally, truncated Ap4A derivatives, such as Some sugar-modified NTPs such as dATP, adenosine(5¢)tetraphospho(5¢)ribose and ddATP and araATP have also been used as ribose(5¢)tetraphospho(5¢)ribose, can be pro- adenylate acceptors yielding dAppppA, duced by ATP N-glycosidase from the marine ddAppppA and araAppppA (24) (Zamecnik et sponge Axilla polypoides. This unusual en- al., 1966; Plateau & Blanquet, 1982; zyme catalyzes hydrolysis of the N-glycosidic Theoclitou et al., 1994; Günther Sillero et al., bond in any compound containing an 1997). It has been shown that dATP and adenosine-5¢-diphosphoryl moiety (Reintamm ddATP, used as the sole NTP in the reaction et al., 2003). In practice, the quantities of mixture, act both as NMP-donors and Ap4A analogs synthesized enzymatically are NMP-acceptors yielding dAppppdA (22) much lower than those obtained by chemical (Randerath et al., 1966; Plateau & Blanquet, procedures. Representative structures of 1982; Günther Sillero et al., 1991; 1997; chemically and enzymatically generated Pietrowska-Borek et al., 2003) and Ap4A analogs are shown in Table 1. ddAppppddA (23)(Günther Sillero et al., 1997), respectively. As mentioned previ- ously, Ap4U can be also formed by yeast ISOTOPICALLY LABELED Ap4A UTP:glucose-1-phosphate uridylyltransferase, ANALOGS AND THEIR APPLICATION which catalyzes transfer of UMP from 14 3 UDP-glucose to ATP (Guranowski et al., [U- C]Ap4Aor[ H]Ap4A, which can either be 2004). Another family of enzymatically pro- produced enzymatically from appropriately la- duced Ap4A analogs, such as Ap4ddA or beled ATP (Jakubowski & Guranowski, 1983; Gp4ddA, can be formed by HIV-1 reverse Baril et al., 1983) or purchased from transcriptase (Meyer et al., 1998). In radiochemical suppliers (Guranowski et al., pyrophosphorolysis-like reactions, this 1983; Guranowski & Blanquet, 1985; transferase carries out nucleotide-dependent Guranowski et al., 1987), have been routinely 958 A. Guranowski 2003 used as substrates for various Ap4A-degrading type of Ap4A derivative to enzymatic cleavage 3 enzymes in quantitative assays. [ H]Ap4A has catalyzed by several specific Ap4A- and/or also been used to study the distribution of Ap4A Ap3A-degrading enzymes (Guranowski et al., binding sites in rat brain (Rodriquez-Pascual et 2003a). Studies of these two sets of Ap4A deriv- al., 1997) and the mechanism by which Ap4A atives also showed that the (asymmetrical)Ap4A mediates stimulation of gluconeogenesis in iso- hydrolases tolerate a bulky substituent such as lated rat proximal tubules (Edgecombe et al., a nucleotide at the 2¢ or 3¢ position of their po- 3 1997). [ H]oAppppoA was used to identify the tential substrates. The (symmetrical)Ap4A Ap4A-binding subunit of calf thymus DNA poly- hydrolase from E. coli also cleaved merase a (EC 2.7.7.7) (Grummt et al., 1979), 3¢-pA(Apppp)A (to ADP and ADP(3¢-pA)) but 3 32 and [ H]Ap4Aand[ P]Ap4A allowed identifi- the yeast Ap4A phosphorylase and Ap3Ahydro- cation of the Ap4A-binding subunit of a lases did not recognize this compound as a sub- multiprotein form of HeLa cell DNA polymer- strate (Guranowski et al., 2003a). ase a (Baril et al., 1983). Ap4A labeled with the stable oxygen isotopes 17O and 18O, 1 4 17 18 (Rp,Rp)-P ,P -bis(5'-adenosyl)-1[ O, O2],4 APPLICATION OF SOME OTHER 17 18 [ O, O2]tetraphosphate, was prepared by Ap4A ANALOGS; THEIR Dixon & Lowe (1989) and used in their elegant INTERACTION WITH DIFFERENT stereochemical analysis of the hydrolytic mech- PROTEINS anism of the (asymmetrical)Ap4A hydrolase from yellow lupin seeds. Another labeled ana- Ap A homologs log, [32P]-P1-(adenosyl-5¢)-P4-(8-azido-adenosyl- 4 ¢ a¢ 32 5 )tetraphosphate ([ - P]8-N3-Ap4A), was syn- So far, Ap5A has been the most useful of the a 32 thesized chemically from [ - P]ATP and Ap4A homologs. Acting as a bisubstrate ana- 8-N3-AMP by the water-soluble carbodiimide log it strongly inhibits adenylate (EC 2.7.4.3) method and used as a photoaffinity label to de- and adenosine (EC 2.7.1.20) kinases. The low- tect Ap4A-binding proteins in cell extracts est Ki values, around 30 nM, were estimated (Prescott & McLennan, 1990; Baxi et al., 1994). for the adenylate kinases from rabbit 32 [b- P]8-N3-Ap4A was also synthesized by (Lienhard & Secemski, 1973) and pig skeletal Chavan & Haley (1994) to study its interaction muscle (Feldhaus et al., 1975). Based on this, with acidic fibroblast growth factor. an Ap5A-Sepharose affinity resin was pre- 3 3 [ H]AppppsAs, (Sp)[H]Ap4AaSand(Rp) pared and successfully used for the isolation of 3 [ H]Ap4AaS, all synthesized enzymatically adenylate kinase from vertebrate muscle 3 from [ H]ATP and the appropriate stereo- (Feldhaus et al., 1975). Ap6Awasamuch a isomers of ATP S, were used as alternative poorer inhibitor, with Ki values of 450 nM for substrates for various Ap4A-degrading enzymes the porcine adenylate kinase (Feldhaus et al., (£a¿ewska & Guranowski, 1990). A 2'-deoxy- 1975) and 55 nM for the rabbit muscle enzyme adenylated derivative of Ap4A labeled on both (Bone et al., 1986b). Co-crystallization of Ap5A adenines, [3H]Apppp[3H]A(2¢-pdA), was used to with adenylate kinases from pig muscle (Pai et demonstrate the cleavage preference of this al., 1977), baker’s yeast (Egner et al., 1987) asymmetric substrate by the (asymmetri- and E. coli (Müller & Schulz, 1988) has allowed 3 cal)Ap4A hydrolases, yielding either [ H]ATP + the three-dimensional structures of these en- 3 ¢ 3 ¢ [ H]AMP(2 -pdA) or [ H]ATP(2 -pdA) + AMP zymes to be studied. The Ki values for Ap5A (Guranowski et al., 2000; Maksel et al., 2001). acting as a competitive inhibitor of MgATP Similarly, AppppA(3¢-[32P]pA) has recently binding were 73 nM and 400 nM, respectively, been synthesized (Günther Sillero et al., 2001) for the adenosine kinase from liver and used to study the susceptibility of this new (Bone et al., 1986b) and bovine adrenal me- Vol. 50 Ap4A analogs 959 dulla (Rotllan & Miras-Portugal, 1985). Ap5A grading enzymes (Jakubowski & Guranowski, and Ap6A also inhibited calf thymus terminal 1983; Plateau et al., 1985; Brevet et al., 1987; deoxynucleotidyl transferase (EC 2.7.7.31) Prescott et al., 1989) and for Ap3A hydrolase with Ki values of 1.5 mM and 1.3 mM, respec- (Barnes et al., 1996). Their use has revealed tively. These two compounds were found to be the asymmetry of the Np4N¢-binding sites of more effective than the diadenosine such enzymes as yeast Ap4A phosphorylase polyphosphates containing 2-, 3- or 4-phos- (Brevet et al., 1987), asymmetrically acting phate groups. However, only Ap5A seems to Ap4A hydrolase from Artemia (Prescott et al., span both the substrate and primer binding 1989), and the human Fhit protein, which is a site domains of the enzyme (Pandey et al., typical dinucleoside triphosphatase (Barnes 1987; Pandey & Modak, 1987). Ap5Aand et al., 1996). In each case, a degree of prefer- Ap6A also inhibit the nucleotide-depleted mito- ential bond cleavage was observed for these chondrial F1-ATPase (EC 3.6.1.34) and have hybrid molecules rather than random degra- been employed in studies of the orientation of dation. For example, phosphorolysis of Ap4G the catalytic and non-catalytic sites of this en- by yeast Ap4A phosphorylase yielded over zyme (Vogel & Cross, 1991). Finally, Ap5A has 7-fold more ATP + GDP than GTP + ADP been shown to inhibit carbamoyl phosphate while hydrolysis of Ap4G by the Artemia synthetase (glutamine hydrolyzing) (EC Ap4A hydrolase yielded a 4.5-fold excess of 6.3.5.5), indicating that this enzyme has two AMP + GTP over GMP + ATP. The Fhit pro- separate binding sites for ATP (Powers et al., tein degraded Ap4G to AMP + GTP (85%) and 1977). GMP + ATP (15%). Ap5A and Ap6A also affect some physiologi- In addition, hybrid Ap4A analogs have been cal processes. Of the various Ap2–6As stud- used as typical bisubstrate analogs in studies ied, Ap5A appeared to be the strongest inhibi- of various nucleoside and nucleotide kinases. tor of ADP-induced human platelet aggrega- Ap4U was shown to be an effective inhibitor tion (Harrison et al., 1977) while both Ap5A of uridine kinase from Ehrlich ascites tumor and Ap6A, which occur naturally in platelets, cells (Cheng et al., 1986) and analogs with N = act as vasopressors (Schlüter et al., 1994). dN were used as probes for distinguishing be- Another naturally occurring homolog of tween kinetic mechanisms of the appropriate Ap4A, Ap3A, is the preferred NpnN substrate kinases: Ap4dT for thymidine kinase (EC for various dinucleoside triphosphatases (EC 2.7.1.21) (Bone et al., 1986a), and Ap4dC, 3.6.1.29) (Lobatón et al., 1975; Jakubowski & Ap4dG and Ap4dA for deoxycytidine (EC Guranowski, 1983; Barnes et al., 1996; Gura- 2.7.1.74), deoxyguanosine (EC 2.7.1.113) and nowski et al., 1996). These specific hydrolas- deoxyadenosine (EC 2.7.1.76) kinases (Ikeda es always liberate AMP (NMP) from their sub- et al., 1986). Ap4dT, Ap5dT and Ap6dT were strates. In addition to Ap3A, they hydrolyze found to be inhibitors of thymidylate kinase Ap4A (to AMP and ATP) and Ap5A, which is (EC 2.7.4.9) from peripheral blast cells of pa- slowly degraded to AMP and p4A (Jaku- tients with acute myelocytic leukemia (Bone 1 bowski & Guranowski, 1983; Brevet et al., et al., 1986b) and Ap5dT and/or P -(5¢-adeno- 1991; Prescott et al., 1992; Barnes et al., syl)-P5-(5¢¢¢-(3¢¢-azido-3¢¢-deoxythymidine)pen- 1996). taphosphate, AZT-p5A, were used in studies of the same kinase from yeast (Lavie et al., 1998a) and E. coli (Lavie et al., 1998b). The Hybrid analogs crystal structures of the latter enzyme Hybrid dinucleoside tetraphosphates, complexed with these compounds have been Ap4Ns (where N A), have been tested as al- solved to 2.0-C and 2.2-C resolution. Davies ternative substrates for different Ap4A-de- and co-workers (1988) tested Ap3dT, Ap4dT, 960 A. Guranowski 2003

Ap5dT and Ap6dT plus their analogs with a Another fluorogenic analog, mAp5mA(26), methylene group a,b to the thymidine resi- was used to measure the binding constants of due, e.g. ApppCH2pdT, as potential inhibitors adenylate kinase–ligand complexes. It was of thymidine kinase, thymidylate kinase and specially designed for the E. coli enzyme, ribonucleotide reductase (EC 1.17.4.1). which has no tryptophan residues and there- Ap5dT was the best inhibitor of the thymidine fore no strong intrinsic fluorescence signal to kinase and both Ap5dT and Ap6dT strongly report ligand binding. Moreover, eAp5A pro- inhibited the thymidylate kinase and were po- duces no significant fluorescence enhance- tent inhibitors of CDP reduction catalyzed by ment with this enzyme, in contrast to mam- the ribonucleotide reductase from L1210 malian cytosolic adenylate kinase. However, cells. 8-Azido-Ap4A(20) was employed to co- mAp5mA produced an exceptionally high fluo- valently label acidic fibroblast growth factor rescence enhancement upon binding to E. coli (FGF-1) (Chavan & Haley, 1994) and sugar- adenylate kinase (about 300%) (Reinstein et modified analogs, dAp4dA and dAp4dT, were al., 1990). shown to be a new type of substrates for sev- eral DNA polymerases of human, bacterial Other Ap A derivatives and viral origin. The strongest activity of 4 those compounds was observed for HIV re- Pyridoxal-tetraphospho-5¢-adenosine (21) verse transcriptase (Victorova et al., 1999). has been used to explore the topography of the catalytic sites of the following enzymes. Acting as an affinity label it modified the ac- Fluoro- and chromogenic analogs tive site of rabbit muscle lactate dehydro- eAp4A and eAp4eA(17) can serve as conve- genase (EC 1.1.1.27) (Tagaya & Fukui, 1986) nient fluorescent substrates of various and glutathione synthetase (EC 6.3.2.3) (Hibi Ap4A-degrading enzymes. Degradation of the et al., 1993) and inactivated rabbit muscle latter results in a 6-fold increase in fluores- adenylate kinase (Yagami et al., 1988) and cence intensity at 410 nm (Wierzchowski et sheep brain pyridoxal kinase (Dominici et al., al., 1985; Rotllán et al., 1991). 5-Bromo- 1988). 4-chloro-3-indolyl-tetraphospho-5¢-adenosine Diinosine polyphosphates (IpnIs) have been (19) was used as a chromogenic substrate for shown to act as selective antagonists at a three different types of Ap4A-degrading en- diadenosine polyphosphate receptor identified zymes in alkaline phosphatase-coupled reac- in rat brain synaptic terminals. The best was 1 tions (Garrison et al., 1993). P -(lin-Benzo- Ip5I, which was 6000-fold more selective for the 5¢-adenosyl)-P5-(5¢-adenosyl)pentaphosphate P4 dinucleotide receptor than for the ATP re- 1 4 and P -(lin-benzo-5¢-adenosyl)-P -(5¢-adeno- ceptor (Pintor et al., 1997). Recently, Ip5Iand syl)tetraphosphate (18), both potent inhibi- Ip6I were proposed as valuable tools for diabe- tors of porcine muscle adenylate kinase, were tes research. They antagonize Ap5A-mediated used to study the active site of this enzyme. inhibition of insulin release from insulin-secret- An increase in fluorescence intensities and ing (INS-1) cells (Verspohl et al., 2003). fluorescence lifetimes of both inhibitors upon binding to adenylate kinase resulted from dis- Ap A analogs modified in the ruption of the intramolecular stacking inter- 4 polyphosphate chain actions that prevail when these ligands are free in solution, suggesting that they bind to Various methylene and halomethylene the enzyme in an “open” or “extended” form analogs of Ap4A have been assayed with spe- (VanDerLijn et al., 1979). cific and non-specific Ap4A-degrading en- Vol. 50 Ap4A analogs 961 zymes, acting as substrates and/or potent in- enzyme. Both sets of analogs were also com- hibitors. Chronologically, the first were petitive inhibitors of E. coli Ap4A hydrolase AppCH2ppA (2), AppCHBrppA, ApCH2pppA with Ki values ranging from 7 mM for App- (3) and ApCH2ppCH2pA (4) (Guranowski et CH2ppA to 250 mM for ApCH2CH2pp- al., 1987). None was hydrolyzed by the (sym- CH2CH2pA. The only disubstituted analog to metrical)Ap4A hydrolase from E. coli but all be hydrolyzed by the E. coli enzyme was were strong inhibitors of this enzyme, with Ki ApCF2ppCF2pA at 0.2% of the rate of Ap4A; values ranging from 3-fold (for ApCH2p-pC- however, several of the bb¢-substituted com- H2pA) to 15-fold (for AppCHBrppA) lower pounds showed a limited degree of asymmet- than the Km for Ap4A (25 mM). The (asymmet- rical cleavage. These results were interpreted rical)Ap4A hydrolase from yellow lupin did in terms of a “frameshift” model for substrate hydrolyze those analogs with one methylene binding in which the oligophosphate chain or halomethylene group. AppCH2ppA com- can position itself in the active site of the en- a b petitively inhibited the hydrolysis of Ap4A zyme with either P or P adjacent to the at- with a Ki 4-fold lower than the Km for Ap4A tacking nucleophile (water) depending on the (0.25 mM versus1mM). The same three electronegativity of the substituent. analogs were substrates for the non-specific Due to their ability to bind tightly to Ap4A phosphodiesterase from yellow lupin. Fi- hydrolases, some of the methylene and nally, of the analogs tested, only ApCH2pppA halomethylene analogs were used to deter- was a substrate of the Ap4A phosphorylase mine the three-dimensional structures of the from yeast. It was degraded 40-fold more enzyme-substrate complexes: a non-degrada- slowly than Ap4A and was also the strongest ble analog with two types of modification, inhibitor of this enzyme, with a Ki of 24 mM ApspCHClppsA(9), was complexed with the versus the Km of 60 mM for Ap4A. Similar enzyme from Lupinus angustifolius (Swar- measurements were subsequently performed brick et al., 2000) and AppCH2ppA with the with the same and other bb'- and hydrolase from Caenorhabditis elegans (Bailey ab,a¢b¢-disubstituted phosphonate analogs of et al., 2002). In physiological assays with Ap4A. AppCF2ppA (5) and AppCCl2ppA were INS-1 cells, ApspCH2ppsA and ApspCHCl- as potent as AppCH2ppA and AppCHBrppA ppsA inhibited insulin release to the same de- as inhibitors of lupin Ap4A hydrolase but gree as Ap4A(Verspohlet al., 2003). were weaker when tested against the E. coli AppCH2ppA, AppCHFppA and AppCHClppA hydrolase (Guranowski et al., 1989). McLenn- were quite potent inhibitors of rat brain an and coworkers (1989) studied a set of 13 adenosine kinase while AppCCl2ppA was ap- phosphonate Ap4A analogs with the (asym- proximately 5-times less potent (Delaney et metrical)Ap4A hydrolase from Artemia and es- al., 1997). Different methylene and halo- tablished that the substrate efficiency of bb' methylene analogs of Ap4A, eAp4A, eAp4eA, -substituted compounds decreased with de- as well as an imido-analog (AppNHppA) were creasing substituent electronegativity examined as effectors of the ADP-ribosylation (O>CF2>CFH>CCl2>CClH>CH2). These com- reaction of histone H1 catalyzed by purified pounds were competitive inhibitors of this en- bovine thymus poly(ADP-ribose)transferase zyme with Ki values that generally also de- (EC 2.4.2.30). Of the compounds tested, creased with electronegativity from 12 mM for ApCH2pppA and eAp4A were shown to be the AppCF2ppA to 0.4 mM for AppCH2ppA (Km most effective inhibitors of the enzyme. The for Ap4A was 33 mM). Disubstituted analogs imido analog was the least effective (Suzuki et were generally less effective inhibitors. How- al., 1987). To the best of my knowledge, there ever, they displayed a low and unexpected are no other reports of effects exerted by the rate of symmetrical cleavage by the Artemia imido analog. 962 A. Guranowski 2003

The monophosphorothioates (Sp)Ap4AaS minal adenosyl residue were replaced with (6)and(Rp)Ap4AaS(7) were tested as alter- sulfur atoms (compound 15) (Walkowiak et native substrates for three specific Ap4A-de- al., 2002). grading enzymes (£a¿ewska & Guranowski, Adenylated derivatives of methanetrisphos- 1990); the asymmetrically degrading Ap4A phonate, so called “supercharged” analogs of hydrolase from yellow lupin seeds, symmet- Ap4A, have been shown to be effective inhibi- rically acting Ap4A hydrolase from E. coli tors of Ap3A hydrolases (Liu et al., 1999). and the Ap4A phosphorylase from yeast. They were, however, poor inhibitors of the hu- Generally, the Rp isomer was a better sub- man and narrow-leafed lupin (asymmetri- strate for all three enzymes than the Sp one. cal)Ap4A hydrolases (Maksel et al., 1999); Interestingly, the Sp analog was cleaved ran- only the triadenylated compound, App-CH- domly by the yeast phosphorylase yielding (ppA)-ppA (10), exerted a significant effect, four reaction products: ATP, ATPaS, ADP with IC50 values estimated in the presence of and ADPaS. Since the latter product re- 50 mMAp4Aof80mM for the human and 40 tained its configuration at the a-phosphorus, mM for the lupin enzyme. this confirmed formation of a covalent A novel family of Ap4A analogs in which NMP-enzyme intermediate as previously adenylate or adenosine-5'-phosphorothioate postulated (Guranowski et al., 1988). Analy- residues are chemically attached to polyols sis of the regiospecificity of diadenosine such as glycerol, erythritol and pentaery- polyphosphate hydrolysis catalyzed by three thritol (Baraniak et al., 1999) have been 18 specific hydrolases using H2 Oand tested in my laboratory as potential sub- ApspppA showed that, for the (symmetri- strates and/or inhibitors of the following en- cal)Ap4A hydrolase from E. coli, attack by zymes: Ap3A hydrolase from yellow lupin water took place only at the b-phosphorus at seeds, human Ap3A hydrolase (Fhit protein), the unmodified end of the molecule, produc- yeast Ap4A phosphorylase, (symmetri- 18 ing [b- O]ADP and unlabeled ADPaS cal)Ap4A hydrolase from E. coli and two (Guranowski et al., 1994). Regardless of (asymmetrical)Ap4A hydrolases, from nar- their configuration, diphosphorothioates row-leafed lupin and humans. All the com- (ApspppsA, 8) were highly resistant to the pounds were resistant to the action of these (asymmetrical)Ap4A hydrolase from Artemia enzymes. However, as inhibitors, they be- while the presence of the P2:P3 methylene haved differently. Generally, the adeno- bridge in ApspCH2ppsA afforded at least a sine-5'-O-phosphorothioylated polyols were further five-fold increase of resistance to hy- much more potent inhibitors of these en- drolysis. Moreover, it was noticed that the zymes than their adenosine-5'-O-phosphory- 1 4 presence of P and P thiophosphates can lated counterparts. Yeast Ap4A phospho- force a symmetrical cleavage of ApspppsA rylase was the most refractory to inhibition. for at least one of the diastereoisomers to The Ap3A hydrolases were inhibited quite ef- yield ADPaS (Blackburn et al., 1987b). An- fectively but the estimated Ki values did not other Ap4A analog with dual modifications, exceed the range of Kms for Ap3A; i.e. they –6 ApspCHClppsA(9), proved to be a promising were not lower than 10 M. The strongest anti-platelet aggregation agent (Chen et al., inhibitory effects were observed for some of 1997). Recently, strong inhibition of these analogs with the Ap4A hydrolases ADP-triggered blood platelet aggregation (Guranowski et al., 2003b): 1,4-di(adeno- was also reported for the analog containing a sine-5'-O-phosphorothio)erythritol (12)ap- central di(hydroxymethyl)phosphonic acid peared to be the strongest inhibitor of the moiety in which both non-bridging oxygens (asymmetrical)Ap4A hydrolase from lupin and of the phosphates linked directly to each ter- humans (Ki values of 0.15 mM and 1.5 mM, re- Vol. 50 Ap4A analogs 963 spectively). Of eight adenosine-5'-O-phospho- differences in the binding sites of these two rylated compounds, the same enzyme was in- similar enzymes. hibited only by 1,4-di(adenosine-5'-O-phos- Of the enzymes that specifically hydrolyze pho)erythritol (11). Di(adenosine-5'-O-phos- dinucleoside tri- or tetraphosphates, only the phorothio), di(phosphorothio)pentaerythritol (symmetrical)Ap4A hydrolase has not yet had (13) and tri(adenosine-5'-O-phosphorothio), its three-dimensional structure determined. thiophosphoro-pentaerythritol (14) were the The above analogs may help this goal to be most powerful inhibitors of the (symmetri- achieved. cal)Ap4A hydrolase from E. coli ever reported (Ki values of 0.04 mM and 0.08 mM, respec- tively). For these two types of enzymes the Ki CONCLUDING REMARKS AND values were lower than the Kms for Ap4A. A PERSPECTIVES comparison of the inhibitory effects exerted by di(adenosine-5'-O-phosphorothio)erythritol As has been shown in this review, Ap4A towards three different (asymmetrical)Ap4A analogs helped to increase our knowledge hydrolases — lupin, human and nematode about the enzymes involved in the metabo- (C. elegans), — showed that each enzyme was lism of Ap4A and other nucleotides. In partic- inhibited to a different extent, probably due ular, the hybrid analogs, Ap4Ns, proved to be to small differences in the shape (topography) alternative substrates of different Ap4A-de- of the active sites of these hydrolases. Inter- grading enzymes. The preference of cleavage estingly, the homologous compound di(adeno- of these asymmetrical compounds as well as sine-5¢-O-phosphorothio)glycerol was not in- other asymmetrically modified Ap4A deriva- hibitory. To further pursue this structure-ac- tives, e.g. 2'- or 3'-adenylylated Ap4As, showed tivity relationship, two di(adenosine-5'- that the active sites of the Ap4A hydrolases O-phosphorothio)diols, 1,4-di(adenosine-5'- and Ap4A phosphorylases are also asymmet- O-phosphorothio)butanediol and 1,5-di(ade- ric. Various Ap4Ns and an Ap4A homolog, nosine-5'-O-phosphorothio)pentanediol, were Ap5A, were applied as useful tools in studies synthesized. These two compounds inhibited of different nucleoside and/or nucleotide kin- neither the lupin nor the human (asymmetri- ases. Ap4A analogs modified in the oligo- cal)Ap4A hydrolase. Thus, one can conclude phosphate chain, particularly the non-degradable that not only the distance between the ones, such as AppCH2ppA and ApspCHClppsA, adenosine-5'-O-phosphorothio-residues mat- form stable complexes with (asymmetri- ters but also the presence of hydroxyls in the cal)Ap4A hydrolases and this allowed the de- chain linking the two nucleosides. termination of the three-dimensional struc- The strongest inhibitors of the (symmetri- tures of these enzymes from the nematode cal)Ap4A hydrolase from E. coli were also the C. elegans (Bailey et al., 2002) and higher strongest when tested with the equivalent plant L. angustifolius (Swarbrick et al., 2000), symmetrically acting enzyme from Salmo- respectively. The very strong inhibition of nella typhimurium, although the Ki values for (symmetrical)Ap4A hydrolases by some adeny- the latter enzyme were markedly higher from lylated and adenosine-5'-phosphorothioated those found for the E. coli counterpart: 0.2 polyols (Guranowski et al., 2003b) encourages mM versus 0.04 mM for di(adenosine-5'-O-phos- one to use these analogs for determination of phorothio), di(phosphorothio)pentaerythritol the three-dimensional structure of a (symmet- and 1.7 mM versus 0.08 mM for tri(adeno- rical)Ap4A hydrolase. a sine-5'-O-phosphorothio), phosphorothio-pen- P -chiral analogs of Ap4A allowed the mecha- taerythritol. This result also points to small nism of Ap4A degradation catalyzed by (asym- 964 A. Guranowski 2003 metrical)Ap4A hydrolase (Dixon & Lowe, 1989) and tetraphosphates. Tetrahedron Lett.; 40: 8603–6. and Ap4A phosphorylase (Łażewskaa& Guranowski, 1990) to be elucidated. Baril E, Bonin P, Burstein D, Mara K, The extracellular, physiological effects of Zamecnik P. (1983) Resolution of the 1 4 ApnAs (Lüthje & Ogilvie, 1988; Miras-Portu- diadenosine 5',5'''-P ,P -tetraphosphate bind- gal et al., 1999; Campbell et al., 1999; Hoyle et ing subunit from a multiprotein form of al., 2001) call for more detailed studies on the HeLa cell DNA polymerase a. Proc Natl interaction of these compounds with pu- Acad Sci U S A.; 80: 4931–5. rine-nucleoside receptors (P1), -mono- Barnes LD, Garrison PN, Siprashvili Z, nucleotide receptors (P2X, P2Y) and specific Guranowski A, Robinson AK, Ingram SW, receptors termed dinucleotide or P4 recep- Croce CM, Ohta M, Huebner K. (1996) Fhit, tors, and on the enzymes located on the cell a putative tumor suppressor in humans, is a 1 3 surface, such as ecto-nucleotide pyrophos- dinucleoside 5',5'''-P ,P -triphosphate phatases/phosphodiesterases, NPP1, NPP2 hydrolase. Biochemistry.; 35: 11529–35. and NPP3 (Vollmeyer et al., 2003). Ap4A Bartkiewicz M, Sierakowska H, Shugar D. analogs should help to discriminate between (1984) Nucleotide pyrophosphatase from po- these proteins and to better understand the tato tubers; purification and properties. Eur 143 role of Ap4A and other NpnN's as signal mole- J Biochem.; : 419–26. cules. Baxi MD, McLennan AG, Vishwanatha JK. Finally, I anticipate studies on new ApnA-de- (1994) Characterization of the HeLa cell rivatives, adenosine(5')polyphospho(5')ri- DNA polymerase a-associated Ap4A binding boses and ribose(5')polyphospho(5'), protein by photoaffinity labeling. Biochemis- that can be produced by the use of a novel en- try.; 33: 14601–7. zyme, ATP N-glycosidase (Reintamm et al., Bessman MJ, Frick DN, O'Handley SF. (1996) 2003). Testing of these truncated Ap4A deriv- The MutT proteins or “Nudix” hydrolases, a atives as potential substrates and/or inhibi- family of versatile, widely distributed, tors of different Ap4A-degrading enzymes will “housecleaning” enzymes. 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