Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide

The dinucleotide carbanicotinamide adenine dinucleotide (carba-NAD), in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide ribonucleoside moiety of NAD, has been synthesized and characterized enzymologically. The synthesis begins with the known 1-aminoribose analogue (+/-)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. The pyridinium ring is first introduced and the resultant nucleoside analogue specifically 5'-phosphorylated. Coupling the racemic carbanicotinamide 5'-mononucleotide with adenosine 5'-monophosphate produces two diastereomeric carba-NAD analogues which are chromatographically separable. Only one diastereomer is a substrate for alcohol dehydrogenase and on this basis is assigned a configuration analogous to D-ribose. The reduced dinucleotide carba-NADH was characterized by fluorescence spectroscopy and found to adopt a "stacked" conformation similar to that of NADH. The analogue is reduced by both yeast and horse liver alcohol dehydrogenase with Km and Vmax values for the analogue close to those observed for NAD. Carba-NAD is resistant to cleavage by NAD glycohydrolase, and the analogue has been demonstrated to noncovalently inhibit the soluble NAD glycohydrolase from Bungarus fasciatus venom at low concentrations (less than or equal to 100 microM).     

Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of beta-NAD.

Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.     

Probing binding requirements of NAD kinase with modified substrate (NAD) analogues

Synthesis of novel NAD(+) analogues that cannot be phosphorylated by NAD kinase is reported. In these analogues the C2' 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 microM) of the human enzyme reported so far.     

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

New coenzymically-active soluble and insoluble macromolecular NAD+ derivatives.

Reaction in dimethyl sulfoxide of nicotinamide 8-bromoadenine dinucleotide with the disodium salt of 3-mercaptopropionic acid afforded nicotinamide-8-(2-carboxyethylthio)adenine dinucleotide, a new NAD+ analogue functionalized at the adenine C-8 position by an omega-carboxylic side chain. Carbodimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexy)-Sepharose) polymers gave the corresponding macromolecular NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine analogue showed a substantial degree of efficiency relative to free NAD+ with yeast alcohol dehydrogenase (47%) but a considerably lower one with rabbit muscle lactate dehydrogenase (3%); the polylysine analogue showed a low degree of efficiency with both enzymes (5-6%).     

Photodependent inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase by (A)-2-azido-NAD+ and (A)-8-azido-NAD

Two photoaffinity analogues of NAD+, (A)-2-azido-NAD+ [nicotinamide 2-azidoadenine dinucleotide] and (A)-8-azido-NAD+ [nicotinamide 8-azidoadenine dinucleotide], have been synthesized, and their reactivities with the rat liver NAD(P)H:quinone acceptor oxidoreductase have been investigated. The reduce nicotinamide nucleotide probes, (A)-2-azido-NADH and (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-2-azido-NAD+ and (A)-8-azido-NAD+ in a photodependent manner, and the inhibition of the enzyme could be prevented by the presence of nicotinamide nucleotide substrates during photolysis. (A)-2-Azido-NAD+ was demonstrated to be a more potent inhibitor than (A)-8-azido-NAD+. In addition, the photodependent inhibition by (A)-8-azido-NAD+ increased when menadione, the substrate of the enzyme, was present during the photolysis, while menadione protected the enzyme from the photodependent inhibition by (A)-2-azido-NAD+. These results indicate that these two NAD+ analogues can be used to identify the nicotinamide nucleotide binding site of this quinone reductase and that they probably bind to the enzyme in different fashions.     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose     

Nicotinamide 2-fluoroadenine dinucleotide unmasks the NAD+ glycohydrolase activity of Aplysia californica adenosine 5'-diphosphate ribosyl cyclase

ADP-ribosyl cyclases catalyze the transformation of nicotinamide adenine dinucleotide (NAD+) into the calcium-mobilizing nucleotide second messenger cyclic adenosine diphosphoribose (cADP-ribose) by adenine N1-cyclization onto the C-1' ' position of NAD+. The invertebrate Aplysia californica ADP-ribosyl cyclase is unusual among this family of enzymes by acting exclusively as a cyclase, whereas the other members, such as CD38 and CD157, also act as NAD+ glycohydrolases, following a partitioning kinetic mechanism. To explore the intramolecular cyclization reaction, the novel nicotinamide 2-fluoroadenine dinucleotide (2-fluoro-NAD+) was designed as a sterically very close analogue to the natural substrate NAD+, with only an electronic perturbation at the critical N1 position of the adenine base designed to impede the cyclization reaction. 2-Fluoro-NAD+ was synthesized in high yield via Lewis acid catalyzed activation of the phosphoromorpholidate derivative of 2-fluoroadenosine 5'-monophosphate and coupling with nicotinamide 5'-monophosphate. With 2-fluoro-NAD+ as substrate, A. californica ADP-ribosyl cyclase exhibited exclusively a NAD+ glycohydrolase activity, catalyzing its hydrolytic transformation into 2-fluoro-ADP-ribose, albeit at a rate ca. 100-fold slower than for the cyclization of NAD+ and also, in the presence of methanol, into its methanolysis product beta-1' '-O-methyl 2-fluoro-ADP-ribose with a preference for methanolysis over hydrolysis of ca. 100:1. CD38 likely converted 2-fluoro-NAD+ exclusively into the same product. We conclude that A. californica ADP-ribosyl cyclase can indeed be classified as a multifunctional enzyme that also exhibits a classical NAD+ glycohydrolase function. This alternative pathway that remains, however, kinetically cryptic when using NAD+ as substrate can be unmasked with a dinucleotide analogue whose conversion into the cyclic derivative is blocked. 2-Fluoro-NAD+ is therefore a useful molecular tool allowing dissection of the kinetic scheme for this enzyme.     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities.

Extracts of Salmonella typhimurium were chromatographed by using Sephadex G-150 to separate the various enzymes involved with pyridine nucleotide cycle metabolism. This procedure revealed a previously unsuspected nicotinamide adenine dinucleotide (NAD) glycohydrolase (EC 3.2.2.5) activity, which was not observed in crude extracts. In contrast to NAd glycohydrolase, NAD pyrophosphatase (EC 3.6.1.22) was readily measured in crude extracts. This enzyme possessed a native molecular weight of 120,000. Other enzymes examined included nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.00), molecular weight of 43,000; NMN glycohydrolase (EC 3.2.2.14), molecular weight of 67,000; nicotinic acid phosphoribosyl transferase (EC 2.4.2.11), molecular weight of 47,000; and nicotinamide deamidase (EC 3.5.1.19), molecular weight of 35,000. NMN deamidase and NMN glycohydrolase activities were both examined for end product repression by measuring their activities in crude extracts prepared from cells grown with and without 10(-5) M nicotinic acid. No repression was observed with either activity. Both activities were also examined for feedback inhibition by NAD, reduced NAD, and NADP. NMN deamidase was unaffected by any of the compounds tested. NMN glycohydrolase was greatly inhibited by NAD and reduced NAD, whereas NADP was much less effective. Inhibition of NMN glycohydrolase was found to level off at an NAD concentration of ca. 1 mN, the approximate intracellular concentration of NAD.     

The crystal structure of complexes between horse liver alcohol dehydrogenase and the coenzyme analogues 3-iodopyridine-adenine dinucleotide and pyridine-adenine dinucleotide.

We have studied the binding of the enzymatically active NAD+ analogue, 3-iodopyridine-adenine dinucleotide, and the inactive analogue, pyridine-adenine dinucleotide to the enzyme horse liver alcohol dehydrogenase using X-ray crystallographic methods. These studies were made under such conditions that crystals of the complexes were isomorphous to apoenzyme crystals. Both analogues bind in the same conformation. The binding of the adenosine moiety is very similar to that of ADP-ribose or NADH bound to the enzyme. The conformation and mode of binding of the remaining portions of the analogue molecules is, however, quite different. The pyridine ring is not situated in the active-site pocket as the nicotinamide group in the isomorphous enzyme-NADH-imidazole complex but lies at the surface of the crevice between the two domains of the subunit, approximately 1.5 nm away from the catalytically active zinc atom. Lys-228 which has been shown to be important for NADH dissociation is in this region of the molecule.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Cofactor-type inhibitors of inosine monophosphate dehydrogenase via modular approach: targeting the pyrophosphate binding sub-domain

Cofactor-type inhibitors of inosine monophosphate dehydrogenase (IMPDH) that target the nicotinamide adenine dinucleotide (NAD) binding domain of the enzyme are modular in nature. They interact with the three sub-sites of the cofactor binding domain; the nicotinamide monophosphate (NMN) binding sub-site (N sub-site), the adenosine monophosphate (AMP) binding sub-site (A sub-site), and the pyrophosphate binding sub-site (P sub-site or P-groove). Mycophenolic acid (MPA) shows high affinity to the N sub-site of human IMPDH mimicking NMN binding. We found that the attachment of adenosine to the MPA through variety of linkers afforded numerous mycophenolic adenine dinucleotide (MAD) analogues that inhibit the two isoforms of the human enzyme in low nanomolar to low micromolar range. An analogue 4, in which 2-ethyladenosine is attached to the mycophenolic alcohol moiety through the difluoromethylenebis(phosphonate) linker, was found to be a potent inhibitor of hIMPDH1 (K(i)=5 nM), and one of the most potent, sub-micromolar inhibitor of leukemia K562 cells proliferation (IC(50)=0.45 μM). Compound 4 was as potent as Gleevec (IC(50)=0.56 μM) heralded as a 'magic bullet' against chronic myelogenous leukemia (CML). MAD analogues 7 and 8 containing an extended ethylenebis(phosphonate) linkage showed low nanomolar inhibition of IMPDH and low micromolar inhibition of K562 cells proliferation. Some novel MAD analogues described herein containing linkers of different length and geometry were found to inhibit IMPDH with K(i)'s lower than 100 nM. Thus, such linkers can be used for connection of other molecular fragments with high affinity to the N- and A-sub-site of IMPDH.     

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.     

Structure of lactate dehydrogenase inhibitor generated from coenzyme.

Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.     

NAD(P)+ analogues: tools for the investigation of the active site of oestradiol 17beta-dehydrogenase from human placenta.

Oestradiol-17beta:NAD+ 17-oxidoreductase from human placenta can accept coenzyme analogues of NAD+ and NADP+ where the amide group is replaced by methyl ketone, nitrile or thioamide. The inhibition with analogues of NAD+ has been studied. The presence of a substituent at C-3 of the pyridinium ring is necessary for the binding. The inhibition by C-4 methylated analogues is very poor, and the effect of a methyl group at C-5 depends on the substituent at C-3. The 1,4,5,6-tetrahydronicotinamide adenine dinucleotide is a competitive inhibitor. Nicotinamide 8-bromoadenine dinucleotide and nicotinamide 8-thioadenine dinucleotide are efficient hydrogen acceptors.     

Cooperativity and noncooperativity in the binding of NAD analogues to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Using NAD analogues as ligands, the structural requirements for negative cooperativity in binding to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase were examined. Although the affinity of nicotinamide hypoxanthine dinucleotide is considerably lower than that of NAD+, it also binds to the enzyme with negative cooperatively. Two pairs of nicotinamide hypoxanthine dinucleotide binding sitess were distinguished, one pair having an affinity for the analogue which is 15 times that of the second pair. Negative cooperativity is also found in the Km values for the analogue. Thus modification of the adenine ring of NAD+ to hypoxanthine does not abolish negative cooperativity in coenzyme binding. Adenosine diphosphoribose binding to the same enzyme shows neither positive nor negative cooperativity, indicating that cooperativity apparently requires an intact nicotinamide ring in the coenzyme structure, under the conditions of these experiments. Occupancy of the nicotinamide subsite of the coenzyme binding site is not necessary for half-of-sites reactivity of alkylating or acylating compounds (Levitzki, A. (1974), J. Mol, Biol. 90, 451-458). However, it can be important in the negative cooperativity in ligand binding, as illustrated by adenosine diphosphoribose which fails to exhibit negative cooperativity. Occupancy of the adenine subsite by adenine is important for stabilization of the enzyme against thermal denaturation. Whether the stabilization is due to an altered conformation of the subunits or stabilization of the preexisting structure of the apoenzyme cannot be determined from these studies. However, nicotinamide hypoxanthine dinucleotide does not contribute to enzyme stability although it serves as a substrate and shows negative cooperativity.     

Selectivity in the binding of NAD(P)+ analogues to NAD- and NADP-dependent pig heart isocitrate dehydrogenases. A nuclear magnetic resonance study.

The coenzyme selectivity of pig heart NAD-dependent and NADP-dependent isocitrate dehydrogenase has been investigated by nuclear magnetic resonance through the use of coenzyme analogues. For both isocitrate dehydrogenases, more than 10-fold lower maximal activity is observed with thionicotinamide adenine dinucleotide [sNAD(P)+] than with NAD(P)+ or acetylpyridine adenine dinucleotide [acNAD-(P)+] as coenzyme. Nuclear Overhauser effect measurements failed to reveal any differences in the adenine-ribose conformations among the enzyme-bound analogues. The 2'-phosphate resonance of the enzyme-bound NADP+ analogues showed the same change in chemical shift observed for the natural coenzyme and revealed the same lack of pH dependence in the range from pH 5.4 to 8.2. NADP-dependent isocitrate dehydrogenase exhibits only small differences in Michaelis constants for the coenzymes with various nicotinamide substituents, reflecting a predominant role for the adenosine moiety in binding. The conformation of the bound nicotinamide-ribose of the natural coenzymes was appreciably different from that of the coenzyme, sNAD(P)+, which shows low catalytic activity. For both isocitrate dehydrogenases, sNAD(P)+ bound to the enzymes exhibits a mixture of syn and anti conformations while only the anti conformation can be detected for NAD(P)+. Chemical shifts of NAD(P)+ enriched with 13C in the carboxamide indicate that interaction of this group with the enzymes may play a role in positioning the nicotinamide ring to participate in catalysis. Our results suggest that, although interaction of the nicotinamide moiety with the enzymes contributes relatively little to the energy of interaction in the binary complex, the enzymes must correctly position this group for the catalytic event.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and structure-activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N(a)-H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N(a)-H or the indole C-2 position influenced the mechanism of action of these analogues. Analogues 68 (IC(50)=10 microM) and 67 (IC(50)=19 microM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC(50)=68 microM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC(50)=11.9 microM), MCF-7 (IC(50)=17.0 microM) and PC-3 (IC(50)=11.1 microM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP.