Dinucleoside Polyphosphate NAD Analogs as Potential NMN Adenylyltransferase Inhibitors. Synthesis and Biological Evaluation

Abstract

Two dinucleoside polyphosphate NAD analogs, P¹-(adenosine-5′)-P³-(nicotinamide riboside-5′)triphosphate (Np₃A, 1) and P¹-(adenosine-5′)-P⁴-(nicotinamide riboside-5′)tetraphosphate (Np₄A, 2), were synthesized and tested as inhibitors of both microbial and human recombinant NMN adenylyltransferase. Compounds 1 and 2 proved to be selective inhibitors of microbial enzymes.     

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.     

Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5'',5''''''-adenosyl) tetraphosphate.

Six new methylenephosphonate analogues of P1P4-bis-(5',5'-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.     

Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.     

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.     

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Novel,potent phenethylamide inhibitors of the hepatitis C virus (HCV) NS3 protease: probing the role of P2 aryloxyprolines with hybrid structures

Synthesis of hybrid HCV NS3 protease/NS4A inhibitors having the 4,4-difluoroaminobutyric acid (difluoroAbu) phenethylamides as P1-P1' and quinolyloxyprolines as P2 fragments led to 7 (IC(50) 54 nM). Molecular modelling suggests that this potent tripeptide inhibitor utilizes interactions in the S1', S1, S2, S3 and S4 sites of the protease.     

Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

Substrate specificity of T4 RNA-ligase. The role of phosphate nucleotide residues in the formation of a covalent AMP-RNA-ligase complex]

The inhibiting effect of adenosine, AMP, ADP, ATP, gamma-thio ATP (I), beta,gamma-imine ATP (II), beta,gamma-methylene ATP (III), P1,P3-di(adenosine-5') triphosphate (IV), P1,P4-di(adenosine-5') tetraphosphate (V) and adenosine 5'-tetraphosphate (VI) on the first step of the T4 RNA ligase reaction was studied. All the compounds tested, with the exception of adenosine, appeared to be competitive inhibitors of the first step of the enzymatic reaction. The inhibition constants (Ki) for the ATP analogs were determined. The data obtained suggest that the efficiency of inhibition depends on the number of phosphate groups and on the structure of ATP analogs. All the compounds under study (I-VI), except for AMP and ADP, form covalent AMP-RNA ligase complexes.     

Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV NS3 protease

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1' region was synthesized and evaluated. The resulting P1-P3 and P2-P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Inhibitory serpins from rye grain with glutamine as P1 and P2 residues in the reactive center

Six of seven serpins detected in grains of rye (Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant (k(a)') for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P2-P1' QQ/S and one with P2-P1' PQ/M were equally efficient inhibitors of chymotrypsin (k(a)' approximately 10(5) M(-1) s(-1)). One serpin with P2-P1' PY/M was a faster inhibitor (k(a)' approximately 10(6) M(-1) s(-1)). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Ostergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.     

Probing pockets S2-S4' of the gamma-secretase active site with (hydroxyethyl)urea peptidomimetics

(Hydroxyethyl)urea peptidomimetics are potent inhibitors of gamma-secretase that are accessible in a few synthetic steps. Systematic alteration of P2-P4' revealed that the corresponding S2-S4' active site pockets accommodate a variety of substituents, consistent with the fact that this protease cleaves a variety of single-pass membrane proteins; however, phenylalanine is not well tolerated at P2'. A compound spanning P2-P3' was identified as a low nM inhibitor of gamma-secretase activity both in cells and under cell-free conditions.     

Inhibition of human napsin A

The newly-discovered human aspartic proteinase, napsin A was not susceptible to protein inhibitors from potato, squash or yeast but was weakly inhibited by the 17 kDa polypeptide from Ascaris lumbricoides and potently by isovaleryl and lactoyl-pepstatins. A series of synthetic inhibitors was also investigated which contained in the P(1)-P(1)' positions the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues and in which the residues occupying P(4)-P(3)' were varied systematically. On this basis, the active site of napsin A can be readily distinguished from other human aspartic proteinases.     

Analogs of diadenosine tetraphosphate (Ap4A)

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in 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.     

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.     

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5'')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.