5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.     

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.     

Membrane permeation characteristics of 5'-modified thymidine analogs.

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.0 mM), the influx rate of each analog was linearly dependent on concentration and insensitive to inhibition by nucleosides, inhibitors of nucleoside transport, and nucleobases. Kinetic analyses using nonlinear regression revealed that a saturable component of 5'-ddThd influx (Km = 200 microM) was competitively inhibited by thymidine (dThd) (Ki = 86 microM) or 5-iodo-2'-deoxyuridine (Ki = 84 microM). Similarly, a saturable component of 5'-N3-5'-ddThd influx (Km = 220 microM) was competitively inhibited by 2-chloroadenosine (Ki = 18 microM). The Ki values for these nucleoside inhibitors were similar to their reported Km values as permeants of the nucleoside transporter. Both 5'-ddThd and 5'-N3-5'-ddThd competitively inhibited the influx of dThd (Km = 60 microM), with similar Ki values (150 and 200 microM, respectively). We conclude that these two 5'-modified dThd analogs enter human erythrocytes both by nonfacilitated diffusion and by the nucleoside transporter. The absence of the 5'-hydroxyl group of dThd (5'-ddThd) resulted in a large increase in the octanol/buffer partition coefficient, in an ability to permeate human erythrocytes by nonfacilitated diffusion, and in a 3-fold diminished binding to the nucleoside transporter. The 5'-azido group (5'-N3-5'-ddThd) resulted in an additional 1.4-fold increase in the octanol/buffer partition coefficient and in a 2-fold increase in the rate of nonfacilitated diffusion.     

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.     

Structure-activity relationship for nucleoside analogs as inhibitors or substrates of adenosine kinase from Mycobacterium tuberculosis. I. Modifications to the adenine moiety

Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado (Ki=0.19 microM), 2-fluoro-Ado (Ki=0.5 microM), 6-cyclopentyloxy-purine riboside (Ki=0.15 microM), and 7-iodo-7-deaza-Ado (Ki=0.21 microM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.     

5'-Deoxy-5'-methylthioadenosine phosphorylase--III. Role of the enzyme in the metabolism and action of 5'-halogenated adenosine analogs

5'-Deoxy-5'-halogenated adenosines are alternative substrates for 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAPase), an enzyme responsible for the metabolism of 5'-deoxy-5'-methylthioadenosine (MTA), a by-product of polyamine biosynthesis. The relative reactivity of these nucleosides with MTAPase from HL-60 human promyelocytic leukemia cells is MTA greater than 5'-deoxy-5'-fluoroadenosine (5'-FlAdo) greater than 5'-chloro-5'-deoxyadenosine (5'-ClAdo) greter than 5'-bromo-5'-deoxyadenosine (5'-BrAdo) greater than 5'-deoxy-5'-iodoadenosine (5'-IAdo). In MTAPase-containing cells, the adenine released from the 5'-halogenated adenosine was incorporated into adenine nucleotide pools; cleavage by (MTAPase appeared to be the rate-limiting step in this process. 5'-BrAdo and 5'-IAdo were growth inhibitors (EC50 values less than 10 microM) of MTAPase-containing cell lines (HL-60 human promyelocytic leukemia and the L5178Y murine lymphoblastic leukemia) but were much less active (EC50 values greater than 65 microM) against MTAPase-deficient cell lines (the CCRF-CEM human T cell leukemia and the L1210 murine leukemia). The full cytotoxicity of these compounds, therefore, appeared to be related to their phosphorolysis by MTAPase. Indirect evidence suggests that 5-halogenated ribose-1-phosphate derivatives of 5'-BrAdo or 5'-IAdo produced by the MTAPase reaction were the active metabolites of these 5'-halogenated adenosines.     

Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase

Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.     

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl-N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2-oxoquinolyl oxy)] -butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 micrograms/ml collagen was 5 microM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 microM, respectively. We purified over 480-fold the soluble low Km form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sepharose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a Km value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 microM respectively, and a Vmax value of 85.3 and 19.8 nmole/min/mg protein, respectively. Ki value of the OPC-13135 for the enzyme was 0.015 microM and was of competitive fashion against cyclic AMP.     

Synthesis and antiproliferative effects of novel 5'-fluorinated analogues of 5'-deoxy-5'-(methylthio)adenosine

5'-Deoxy-5'-[(monofluoromethyl)thio]adenosine (9) and 5'-deoxy-5'-fluoro-5'-(methylthio)adenosine (10), two novel analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), have been synthesized and evaluated for their substrate and inhibitory activities toward MTA phosphorylase and for their biological effects in L1210 (MTA phosphorylase deficient) and L5178Y (MTA phosphorylase containing) murine leukemia cell lines. Compound 9 was a potent competitive inhibitor of MTA phosphorylase with a Ki value of 3.3 microM and was also a substrate, with activity approximately 53% that of MTA. Compound 10 was significantly less inhibitory toward the phosphorylase with a Ki value of 141 microM; its lack of substrate activity was attributed to rapid nonenzymatic degradation. The 50% growth inhibitory concentrations (48 h) of 9 were 300 and 200 microM in L1210 and L5178Y cells, respectively; for 10, these respective values were 2 and 0.7 microM. The initial characterization of 9 in these systems reveals that it differs from MTA by not acting as a product regulator of the polyamine biosynthetic pathway.     

Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6

S-(-)-Nicotine and 13 of the most prevalent nicotine-related alkaloids and metabolites (i.e., S-(-)-nornicotine, myosmine, beta-nicotyrine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-anabasine, S-(-)-N-methylanabasine, anabaseine, S-(-)-anatabine, nicotelline, and 2,3'-bipyridyl) were evaluated as inhibitors of human cDNA-expressed cytochrome P-450 2A6 (CYP2A6) mediated coumarin 7-hydroxylation. Tobacco alkaloids myosmine, S-(-)-nornicotine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-N-methylanabasine, anabaseine, and nicotelline had Ki values for inhibition of coumarin 7-hydroxylation ranging from 20 microM to more than 300 microM whereas nicotine and S-(-)-anatabine were much more potent (i.e. 4.4 and 3.8 microM, respectively). The tobacco alkaloids 2,3'-bipyridyl (7.7 microM) and S-(-)-anabasine (5.4 microM), were somewhat less potent compared with S-(-)-nicotine or S-(-)-anatabine in inhibition of human CYP2A6. beta-Nicotyrine, in which the N-methylpyrrolidino moiety of nicotine was replaced by the aromatic N-methylpyrrole ring, was shown to inhibit human CYP2A6 with much greater potency (Ki=0.37 microM) compared with S-(-)-nicotine. Among the compounds examined, only nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6. The potency of the mechanism-based CYP2A6 inhibitors suggests that, for smokers, modulation of CYP2A6 may be greater than that predicted on the basis of serum concentration of these alkaloids. Our results indicate that the prominent nicotine-related alkaloid beta-nicotyrine present after smoking potently inhibits human CYP2A6.     

Conformational and steric aspects of phenylethanolamine and phenylethylamine analogues as substrates or inhibitors of phenylethanolamine N-methyltransferase

The conformational and steric aspects of binding to phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) for phenylethanolamine substrates and phenylethylamine inhibitors were probed with three conformationally defined analogues (11, 12, and 13) of phenylethylamine (1) and phenylethanolamine (6) containing the benzobicyclo[3.2.1]octane skeleton. The 2-aminotetralin (2AT) moiety in conformationally defined analogues 11, 12, and 13 exists in a half-chair conformation with an equatorial amino group. Although conformationally restricted phenylethylamine analogue 2AT (3, Ki = 6.8 microM) and conformationally restricted phenylethanolamine analogues (cis)- and (trans)-2-amino-1-tetralol (9, Km = 22 microM; Vmax = 0.15; 100 X Vmax/Km = 0.68; 10, Ki = 9.4 microM) are good ligands for PNMT, none of the analogues 11, 12, and 13 showed activity as a substrate of PNMT. The fact that 11 (Ki = 206 microM) is more potent than analogues 4 (Ki = 1296 microM) and 5 (Ki = 479 microM), with a half-boat 2AT moiety, suggests that PNMT preferentially binds the half-chair conformation of 2AT at the active site. This is consistent with previous findings that a fully extended conformation for the aminoethyl side chain of phenylethylamine inhibitors is optimal for PNMT binding. The reduced activity of 11, 12 (Ki = 1246 microM), and 13 (Ki = 3000 microM), compared with 2AT and (cis)- and (trans)-2-amino-1-tetralol (9 and 10) is consistent with a negative steric interference from the extra ethano bridge in 11, 12, and 13. The results from 11, 12, and 13, combined with previous findings, suggest that PNMT interacts better with relatively planar ligands.     

5'-Methylthioadenosine phosphorylase-L. Substrate activity of 5'-deoxyadenosine with the enzyme from Sarcoma 180 cells

5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μM units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem. Pharmac. 27, 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K_i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K_m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K_m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K_m for 5′-dAdo is 23 μM (V_max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.     

Inhibition of human liver folylpolyglutamate synthetase by non-gamma-glutamylatable antifolate analogs

Folylpolyglutamate synthetase (FPGS) catalyzes the gamma-glutamylation of both folates and folate antagonists and has been found to be essential for the survival of mammalian cells. Twelve analogs of the antifolates aminopterin (AMT) and methotrexate (MTX) having the -(CH2)2COOH moiety replaced by -(CH2)nX, where X = SO3H,PO3H2 or NH2, were evaluated as inhibitors of FPGS isolated from human liver. The AMT analogs were consistently found to be better inhibitors than their MTX counterparts, following the order of Km values determined for the parent antifolates as FPGS substrates. For the amino and phosphonate (but not for the sulfonate) compounds, inhibitory efficiencies were markedly dependent on the methylene chain length, with the most effective inhibitors having the groups -(CH2)3NH2(Ki = 0.2 microM) and -(CH2)2PO3H2 (Ki = 1.9 microM). Of those compounds exhibiting Ki values less than 200 microM, six were competitive inhibitors whereas three showed mixed inhibition (Ki' = approximately 6 Ki) when analyzed using AMT as the variable substrate. This demonstration of mixed inhibition of FPGS is consistent with the binding of inhibitor to a second site on the enzyme. Very similar Ki values (0.2-0.3 microM) were obtained for the -(CH2)3NH2 analog of AMT when using folic acid, AMT, MTX, and gamma-glutamyl-MTX as variable substrates, suggesting that the same enzymatic site on FPGS is active in the gamma-glutamylation of these four folyl derivatives. These findings serve to identify structural features which are important for inhibition of human liver FPGS and may therefore prove useful for the design of new compounds having potential as chemotherapeutic agents.     

Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase

The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L.mmol-1.cm-1) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (-)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (-)-psi-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (-)-cocaine was a poor substrate for debenzoylation, it was a fairly good competitive inhibitor (Ki approximately 10 microM) of the hydrolysis of other substrates. The cocaine metabolites (-)-norcocaine, (-)-benzoylecgonine and (-)-ecgonine methyl ester inhibited BChE with Ki values of 15, 76 and 1300 microM, respectively. (+)-psi-Cocaine had Ki = 3 microM, p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (-)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar Ki values (160-170 microM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.     

Conformational preference for the binding of biaryl substrates and inhibitors to the active site of phenylethanolamine N-methyltransferase

We have previously described regions of steric bulk tolerance in the aromatic-ring binding site of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) for phenylethanolamine substrates and alpha-methylbenzylamine inhibitors. For bound substrates, this region is located in the vicinity of the para position of the aromatic ring, while for bound alpha-methylbenzylamine inhibitors, it is located in the region complementary to the meta position. In the present study, we sought to determine the preferred conformation of the biaryl portion of (m-phenylphenyl)- and (p-phenylphenyl)ethanolamine (4 and 5, respectively) as well as for m-phenyl- and p-phenyl-alpha-methylbenzylamine (7 and 8, respectively) for PNMT active site interactions. Planar derivatives of 4, 5, 7, and 8 were obtained through the synthesis of 2-(1-fluorenyl)-2-hydroxyethylamine (9), 2-(2-fluorenyl)-2-hydroxyethylamine (10), 1-(1-fluorenyl)ethylamine (11), and 1-(2-fluorenyl)ethylamine (12). The four fluorene derivatives were examined for in vitro activity as substrates and inhibitors of the PNMT-catalyzed reaction. As in the case of 4, 5, 7, and 8, we have observed a positional preference for the alkylamine side chain with respect to the biphenyl skeleton present in 9-12. Thus, fluorenylethanolamine 10 ("p-biphenyl") displays a Michaelis constant (Km = 26 microM) that is approximately 10 times lower than that for 9 ("m-biphenyl", Km = 297 microM); in the alpha-methylbenzylamine inhibitors, fluorenyl derivative 11 ("m-biphenyl", Ki = 4.14 microM) is approximately 40 times better than 12 ("p-biphenyl", Ki = 185 microM) for in vitro inhibition of PNMT. In each case, conformational restriction of the biaryl system present in 4, 5, 7, and 8, such that the aromatic rings are coplanar, resulted in enhanced affinity for the PNMT active site. Thus, conformational restriction of ethanolamine 5 (Km = 82 microM) as in 10 (Km = 26 microM) and alpha-methylbenzylamine 7 (Ki = 89 microM) as in 11 (Ki = 4.14 microM) leads, in each case, to a stronger enzyme-ligand dissociable complex. These results, in conjunction with others from these laboratories, indicate that the PNMT active site beyond the zone that interacts with the central aromatic ring portion of phenylethanolamine substrates and alpha-methylbenzylamine inhibitors is essentially a flat, hydrophobic pocket.     

Trypanosoma cruzi adenine nucleoside phosphorylase. Purification and substrate specificity

An adenine nucleoside phosphorylase has been partially purified from extracts of epimastigotes of the Peru strain of Trypanosoma cruzi, the causative agent of Chagas' disease. The purification procedure separated this enzyme from the three other nucleoside-cleaving enzymes found in extracts. The adenine nucleoside phosphorylase, which efficiently cleaved 5'-deoxy-5'-methylthioadenosine (MTA), had a particle weight of 68,000 and exhibited a broad pH optimum between pH 6 and 8. In addition to MTA, the purified enzyme cleaved and synthesized adenosine and 2'-deoxyadenosine with high efficiency. This contrasts to the enzyme from S-180 cells which has been reported to cleave adenosine poorly and not to cleave 2'-deoxyadenosine. Several observations suggested that the three substrates, MTA, adenosine and 2'-deoxyadenosine, use a common catalytic site: (a) all served as alternate-substrate inhibitors exhibiting mutually competitive inhibition with Ki values equivalent to their respective Km values, (b) 5'-chloroformycin A exhibited a competitive Ki value of 4 microM with each nucleoside substrate, and (c) the Km value of phosphate derived from initial velocity studies (180 +/- 20 microM) was independent of the nucleoside substrate. Substrate specificity studies in both the synthesis and cleavage direction indicated that the enzyme had a broad specificity for bases and nucleosides. For the synthesis of nucleosides, the enzyme demonstrated a preference for an amino group in the position equivalent to the 6 position of purine. Compounds containing a hydroxyl group in this position were not substrates. Although a hydrogen or methyl group could substitute for a 6-amino group, a marked decrease in substrate efficiency was observed with these compounds. Alterations in the purine ring led to decreases in the maximal velocity values as evidenced by the substrate or nonsubstrate properties of 1-, 3-, and 7-deazaadenine and 4-aminopyrazolo[3,4-d]pyrimidine. The Km values for 5-methylthioribose 1-phosphate, ribose 1-phosphate and 2'-deoxyribose 1-phosphate with adenine serving as acceptor were 21, 150 and 370 microM. For nucleoside cleavage, the T. cruzi enzyme catalyzed the phosphorolysis of a variety of 5'-substituted adenine-containing nucleosides including those possessing 5'-hydrogen-, hydroxyl-, halogeno-, alkylthio-, amino- and azido-moieties. Inclusion of an ionized group in the 5'-position, such as 5'-carboxy-5'-deoxyadenosine or AMP, precluded substrate activity. 3'-Deoxyadenosine, arabinosyladenine and alpha-adenosine did not serve as substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Codeine O-demethylation: rat strain differences and the effects of inhibitors

The oxidative metabolism of more than 20 drugs (e.g. sparteine, debrisoquine, dextromethorphan) is mediated by cytochrome P450IID6. Codeine O-demethylation to morphine was recently demonstrated to co-segregate with the polymorphic metabolism of debrisoquine and dextromethorphan. The female Dark-Agouti rat (DA) is an animal model for the poor metabolizer phenotype (PM) using debrisoquine or dextromethorphan as substrates. Studies were carried out to evaluate codeine metabolism in liver microsomes from female DA and Sprague-Dawley (SD) rats. The intrinsic clearance of codeine to morphine was 10-fold lower in DA rats due to a 5-fold higher Km (287 vs 49 microM) and a 2-fold lower Vmax (48 vs 94 nmol/mg/hr). Nineteen drugs were tested for inhibition of codeine O-demethylation. The four most potent competitive inhibitors were dextromethorphan (Ki = 2.53 microM), propafenone (Ki = 0.58 microM), racemic methadone (Ki = 0.3 microM) and quinine (Ki = 0.07 microM). The differences in morphine formation from codeine between SD and DA rats and the inhibition results show that this animal model appears to be a suitable model for the human EM and PM phenotypes, respectively. These strains could be used to study the pharmacodynamic consequences of the genetic polymorphism in codeine O-demethylation, and the effects of metabolic inhibitors. The outcome of these studies could impact on the therapy of pain control.     

New isoflavonoids as inhibitors of porcine 5-lipoxygenase.

The inhibitory activity of new isoflavonoids on 5-lipoxygenase of porcine leukocytes was investigated. Isoflavans (I) proved to be stronger inhibitors than isoflavones (II). The isoflavans containing ortho-hydroxy groups in ring A showed the lowest Ki values (0.8-50 microM). In comparison, isoflavans with meta-dihydroxy groups exhibited Ki values higher than 150 microM. The effect of commercial antioxidants was tested also on porcine 5-lipoxygenase. Butylated hydroxyanisole (Ki: 25 microM) and butylated hydroxytoluene (Ki: 55 microM) revealed moderate inhibitory activity, whereas L-ascorbic acid, L-ascorbyl palmitate, dl-alpha-tocopherol and n-propyl gallate showed weak inhibitory activities (Ki: 100-260 microM).     

Regioselective 2-hydroxylation of 17beta-estradiol by rat cytochrome P4501B1

Previous work demonstrated that human cytochrome P4501B1 (CYP1B1) forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Here, we present results from kinetic studies characterizing the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using 17beta-estradiol (E2) as a substrate. Km and Kcat values were estimated using the Michaelis-Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61+/-0.23 and 1.84+/-0.73 microM; the turnover numbers (Kcat) were 0.23+/-0.02 and 0.46+/-0.05 pmol/min/pmol P450; and the catalytic efficiencies (Kcat/Km) were 0.37 and 0.25, respectively. For human CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 1.22+/-0.25 and 1.10+/-0.26; the turnover numbers were 1.23+/-0.06 and 0.33+/-0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. The turnover number ratio of 4- to 2-hydroxylation was 3.7 for human CYP1B1 and 0.5 for rat CYP1B1. These results indicate that, although rat CYP1B1 is a low Km E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3',5'-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 microM, respectively. The Ki values of 7,8-benzoflavone (alpha-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 microM, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.     

5-Benzylacyclouridine and 5-benzyloxybenzylacyclouridine,potent inhibitors of uridine phosphorylase

Various pyrimidine acyclonucleosides (l-(2'-hydroxyethoxymethyl)uracils) are specific inhibitors of uridine phosphorylase [Niedzwicki et al., Biochem. Pharmac.30, 2097 (1981)]. 5-Benzyluracils have also been shown to inhibit this enzyme [Baker and Kelley. J. med. Chem.13, 461 (1970); Woodman et al., Biochem. Pharmac. 29,1059 (1980)]. We have synthesized the acyclonucleoside analogs of 5-benzyluracil (BU) and 5-benzyloxybenzyluracil (BBU). These compounds, 5-benzyl-1-(2'-hydroxy-ethoxymethyl) uracil (BAU) and 5-(m-benzyloxybenzyl)-1-(2'-hydroxyethoxymethyl)uracil (BBAU), are potent inhibitors of uridine phosphorylase. K_i values of 98 and 32 nM were estimated for BAU and BBAU respectively. These compounds are better inhibitors of uridine phosphorylase than BU (K_i = 1575 nM), BBU (K_i = 270 nM), and all other compounds previously tested, and they have no effect on thymidine phosphorylase, uridine-cytidine kinase, or thymidine kinase. Potential chemotherapeutic applications of BAU and BBAU are discussed.