Thiazole-4-carboxamide Adenine Dinucleotide Research Articles

Abstract 1506: Prodrug targeted gene therapy for colorectal cancer

Abstract In the US colorectal cancer is the second and third most common cause of cancer death in men and women, respectively. The drawback of conventional therapy is its non-specificity and a median survival of only 10-15 months with the present combinations. Our studies with human colorectal cancer cell lines demonstrate a relationship between human nicotinamide mononucleotide adenylyltransferase-2 (hNMNAT-2) expression and cell kill with tiazofurin (TR) and benzamide riboside (BR). Thus, the purpose of the present study is to examine whether over-expressing hNMNAT-2 selectively confers sensitivity to TR and BR in human colorectal cancer cells that show resistance to TR and BR therapy. TR and BR are pro-drugs that are metabolized by the action of NMNAT to its active form, TAD (thiazole-4-carboxamide adenine dinucleotide) and BAD (Benzamide adenine dinucleotide), respectively. TAD/BAD potently inhibit inosine-5’-monophosphate dehydrogenase (IMPDH) causing reduced guanylate synthesis resulting in cancer cell death. We achieved 2- to 5-fold increase in expression of hNMNAT-2 by transfecting colorectal cancer cells (Caco-2 and HT-29) with pTracerNMNAT-2 as demonstrated by Western blot and FACS. hNMNAT-2 transfected colorectal cancer cells exhibited a 3- to 7-fold increase in tumor-cell kill with TR and BR. hNMNAT-2 transfected cells did not exhibit modulation of either NAD levels or IMPDH expression. Folate receptor alpha (FR-α) is overexpressed on the colorectal cancer cell surface to sequester available folate to promote cancer cell proliferation. Primary colorectal carcinomas which exhibit high FR-α overexpression also correlated with decreased 5-year survival. We demonstrated an upregulation of FR-α expression in human colorectal cancer cells (up to 10-fold) by adaptation to growth in physiological low folate medium resulting in 2- to 5-fold increase in colorectal cancer cell-kill with TR or BR encapsulated in folate-tethered liposomes. To translate in vitro studies to in vivo applicability, we are utilizing adeno-associated virus (AAV-2) to transduce hNMNAT-2 gene into colorectal cancers implanted in athymic mice. To achieve this goal, we engineered hNMNAT-2 gene into AAV-2 vector to increase efficiency of gene transfer, and encapsulated in folate-tethered liposomes to target colorectal cancer cells. The potential significance of these new proof-of principle studies is that they will set the stage for the next level of studies that could culminate in a new form of pro-drug targeted gene therapy for colorectal cancer with enhanced tumor cell-kill with little toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1506.

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2′-MeAD ( 1) and T-3′-MeAD ( 2) containing, respectively, a methyl group at the ribose 2′- C-, and 3′- C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2′- C-methyl- or 3′- C-methyl-adenosine 5′-monophosphate with 2′,3′- O-isopropylidene-tiazofurin 5′-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2′-MeAD and T-3′-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3′-MeAD was comparable to that of parent compound TAD, while T-2′-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2′-MeAD and T-3′-MeAD were found to inhibit the growth of K562 cells (IC 50 30.7 and 65.0 μM, respectively).

Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.

Nucleoside and Non-nucleoside IMP Dehydrogenase- Inhibitors as Antitumor and Antiviral Agents

IMP dehydrogenase (IMPDH) is an enzyme which catalyzes the NAD-dependent conversion of inosine 5 -monophosphate (IMP) to xanthosine 5 -monophosphate (XMP) at the metabolic branch point in the de novo purine nucleotide synthetic pathway. IMPDH was shown to be increased significantly in cancer cells and therefore considered to be a sensitive target for cancer chemotherapy. By blocking the conversion of IMP to XMP, IMPDH inhibitors lead to depletion of the guanylate (GMP, GDP, GTP and dGTP) pools. Two isoforms of human IMPDH, designed type I and type II, have been identified and sequenced. Type I is constitutively expressed and is the predominant isoform in normal cells, while type II is selectively up-regulated in neoplastic and replicating cells. Two types of IMPDH inhibitors, endowed with antineoplastic, antiviral and immunosoppressive activity, have been discovered so far: nucleoside inhibitors, such as ribavirin and tiazofurin, and non-nucleoside, such as mycophenolic acid. Ribavirin produces IMPDH inhibition via its anabolite 5 -monophosphate. Tiazofurin inhibits the enzyme after metabolic conversion into thiazole-4-carboxamide adenine dinucleotide (TAD), an analogue of the cofactor NAD. It was hypothesized that the inhibitory activity of tiazofurin is due to an attractive electrostatic interaction between the heterocyclic sulphur atom and the furanose oxygen 1 which constrain rotation about the C-glycosidic bond in tiazofurin and in its active anabolite TAD. To check this hypothesis, we studied several C-nucleosides related to tiazofurin and their NAD analogues. Non-nucleoside IMPDH inhibitors are also reviewed.

Synthesis of nonhydrolyzable analogues of thiazole-4-carboxamide and benzamide adenine dinucleotide containing fluorine atom at the C2' of adenine nucleoside: induction of K562 differentiation and inosine monophosphate dehydrogenase inhibitory activity.

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

Potent inhibitors of human inosine monophosphate dehydrogenase type II. Fluorine-substituted analogues of thiazole-4-carboxamide adenine dinucleotide.

Three analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) (1-3) containing a fluorine atom at the C2' of the adenine nucleoside (in the ribo and arabino configuration) and at the C3' (in the ribo configuration) were synthesized in high yield from the corresponding 5'-monophosphates of 2'-deoxy-2'-fluoroadenosine (9), 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (17), and 3'-deoxy-3'-fluoroadenosine (14), respectively. Pure 2',3'-O-isopropylidene-tiazofurin 5'-phosphorimidazolide (8) was obtained by phosphorylation of the protected tiazofurin followed by treatment with carbonyldiimidazole and HPLC purification. Reaction of 8 with 9 in DMF-d7 (monitored by 1H and 31P NMR) afforded the desired dinucleotide 12, which after deisopropylidenation gave 1 in 82% yield. Small amounts of symmetrical dinucleotides AppA (10, 7.2%) and TRppTR (11, 8.0%) were also isolated during HPLC purification of the major product 12. In a similar manner, compounds 2 and 3 were obtained by coupling of 8 with 14 and 17 in 80% and 76% yield, respectively. All newly prepared fluoro-substituted compounds as well as beta-CF2-TAD, earlier synthesized by us, showed good inhibitory activity against inosine monophosphate dehydrogenase type II, the isozyme which is predominant in neoplastic cells. Binding of 1 (Kis = 0.5 microM), 2 (Kis = 0.7 microM), and 3 (Kis = 2.9 microM) was comparable to that of TAD (Ki = 0.2 microM). The difluoromethylene bisphosphonate analogue, beta-CF2-TAD (Ki = 0.17 microM), was found to be equally effective as the best cofactor-type inhibitor, beta-CH2-TAD (Ki = 0.11 microM). Interestingly, the level of inhibition of horse liver alcohol dehydrogenase by these compounds was found to be much lower (0.1 mM for 1 and 2 and no inhibition up to 10 mM for 3). These findings show that inhibition of tumor-induced inosine monophosphate dehydrogenase type II is selective and may be of therapeutic interest.

Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 μM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 μM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8- 3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogynase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 μM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.

Crystallographic studies of two alcohol dehydrogenase-bound analogues of thiazole-4-carboxamide adenine dinucleotide (TAD), the active anabolite of the antitumor agent tiazofurin.

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the antitumor drug tiazofurin. Beta-methylene TAD (beta-TAD) is a phosphodiesterase-resistant analogue of TAD, active in tiazofurin-resistant cells. Beta-methylene SAD (beta-SAD) is the active selenium derivative of beta-TAD. Both agents are analogues of the cofactor NAD and are capable of acting as general dehydrogenase inhibitors. Crystal structures of beta-TAD and beta-SAD bound to horse liver alcohol dehydrogenase (LADH) are presented at 2.9 and 2.7 A, respectively. Both complexes crystallize in the orthorhombic space group C222(1) and are isomorphous to apo-LADH. Complexes containing beta-TAD and beta-SAD were refined to crystallographic R values of 15% and 16%, respectively, for reflections between 8 A and the minimum d spacing. Conformations of both inhibitors are similar. beta-TAD and beta-SAD bind to the "open" form of LADH in the normal cofactor-binding cleft between the coenzyme and catalytic domains of each monomer. Binding at the adenosine end of each inhibitor resembles that of NAD. However, the positions of the thiazole and selenazole heterocycles are displaced away from the catalytic Zn cation by approximately 4 A. Close intramolecular S-O and Se-O contacts observed in the parent nucleoside analogues are maintained in both LADH-bound beta-TAD and beta-SAD, respectively. These conformational constraints may influence the binding specificity of the inhibitors.

Synthesis of thiazole-4-carboxamide-adenine difluoromethylenediphosphonates substituted with fluorine at C-2′ of the adenosine

Synthesis of an analogue 3 of thiazole-4-carboxamide adenine-dinucleotide (TAD) in which the α-oxygen atom of the pyrophosphate bridge is replaced by a difluoromethylene group has been achieved. Likewise, 2′-deoxy-2′-fluoroadenosine containing analogues of TAD ( 4) and its difluoromethylenediphosphonate congener ( 5 have been synthesized. Adenosine 5′-difluoromethylenediphosphonate ( 8) was prepared from 5′- O-tosyladenosine ( 6) and tris-tetra- n-butylammonium)difluoromethylene-diphosphonate ( 7) by a modified procedure of Poulter's. 2 Compound 8 was converted into the 2′-3′-cyclic carbonate 9 by treatment with triethyl orthoformate. Treatment of 9 with 2′-3′- O-isopro-pylidenetiazofurin ( 10) in pyridine in the presence of DCC gave a mixture of dinucleotide 11 and the isopropylidene-protected diadenosine tetraphosphonate 12. After deprotection of 11, the desired β-difluoromethylene TAD ( 3_ was separated by HPLC as the minor product. The diadenosine tetraphosphonate 12, an analogue of Ap 4A, was obtained as the major component. Alternatively, 2′-3′- O-isopropylidenetiazofurin ( 10) was tosylated, and the product 13 was further converted into the corresponding difluoromethylenediphosphonate 14 by coupling with 7. DCC-catalyzed coupling of 14 with 2′-deoxy-2′-fluoroadenosine ( 15) followed by deisopropylidenation afforded the anlogue 5. Again the corresponding tetraphosphonate analogue of tiazofurin 17 was the predominant product. Dinucleotide 4 was obtained by coupling of the carbonyldiimidazole-activated tiazofurin 5′-monophosphate with 2′-deoxy-2′-fluoroadenosine 5′-monophosphate. 2′-Deoxy-2′-fluoroadenosine (′15) was prepared efficiently from the known N 6-benzoyl-3′- O-tetrahydropyranyladenosine ( 18), which was converted into 3′- O-tetrahydropyranyl-2′- O-triflyl-5′- O-trityladenosine ( 20) by tritylation and triflation. Treatment of 20 with sodium acetate in hexamethylphosphoric triamide, followed by deaceltylation afforded 9-(3- O-tetrahydropyranyl-5- O-trityl-β- d-arabinofuranosyl)- N 6-benzoyladenine ( 22), which was then treated with DAST. After deprotection of the product, 15 was obtained in good yield.

Synthesis of thiazole-4-carboxamide adenine dinucleotide (TAD) analogues with an altered anhydride bridge

Synthesis of thiazole-4-carboxamide adenine dinucleotide (TAD) analogues with an altered anhydride bridge

Cytotoxicity of tiazofurin and its arabinose and xylose analogues in K562 cells

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.

Induction of HL60 Cell Differentiation by Tiazofurin and Its Analogues: Characterization and Efficacy

Among inducers of myeloid differentiation for leukemic cells, tiazofurin is of special interest because its mechanism of action is known; it inhibits inosine monophosphate dehydrogenase and thus decreases the guanine nucleotide pool. Reported here are three aspects of tiazofurin induction of myeloid differentiation in HL60 human acute promyelocytic leukemia cells. First, inductive efficacy was evaluated for analogues ara-tiazofurin, xylo-tiazofurin, and selenazofurin, for dinucleotide anabolites thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD), and for a phosphodiesterase-resistant TAD analogue, beta-methylene TAD. The results showed that the parent compounds are more effective inducers than the dinucleotide derivatives and that the selenazole analogues are more effective inducers than the thiazole compounds. Second, HL60 cell induction by tiazofurin was shown to be synergistic with that produced by the antiviral agent ribavirin. Finally, tiazofurin was found to induce expression of a phosphatidylinositol-specific phospholipase C-sensitive Fc gamma-receptor III (FcRIII) on HL60 cells, a feature consistent with neutrophilic, but not monocytic, differentiation.

Induction of HL60 cell differentiation by tiazofurin and its analogues: characterization and efficacy

Among inducers of myeloid differentiation for leukemic cells, tiazofurin is of special interest because its mechanism of action is known; it inhibits inosine monophosphate dehydrogenase and thus decreases the guanine nucleotide pool. Reported here are three aspects of tiazofurin induction of myeloid differentiation in HL60 human acute promyelocytic leukemia cells. First, inductive efficacy was evaluated for analogues ara-tiazofurin, xylo-tiazofurin, and selenazofurin, for dinucleotide anabolites thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD), and for a phosphodiesterase-resistant TAD analogue, beta-methylene TAD. The results showed that the parent compounds are more effective inducers than the dinucleotide derivatives and that the selenazole analogues are more effective inducers than the thiazole compounds. Second, HL60 cell induction by tiazofurin was shown to be synergistic with that produced by the antiviral agent ribavirin. Finally, tiazofurin was found to induce expression of a phosphatidylinositol-specific phospholipase C- sensitive Fc gamma-receptor III (FcRIII) on HL60 cells, a feature consistent with neutrophilic, but not monocytic, differentiation.

Tiazofurin Down-Regulates Expression of c-Ki-ras Oncogene in a Leukemic Patient

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Determination of thiazole-4-carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of ISpmol and linearity up to 3nmol. The coefficient of variation was 0.6 ± 0.1% for retention time and 2 ± 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m 2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 μM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m 2 (21.1 mM) were 42.8, 26.1, and 1.4 μM respectively.

Dehydrogenase binding by tiazofurin anabolites

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the K m values for IMP and NAD were 29 and 54 μM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5′-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the K i values of RMP (0.58 μM) and TAD (0.075 μM) were several orders of magnitude lower than those of XMP (85 μM) and NADH (94 μM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.

IMP dehydrogenase: Inhibition by the anti-leukemic drug, tiazofurin

Tiazofurin through its active metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) inhibits IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis. IMP dehydrogenase activity in human leukemic cell extracts (33.4 ± 0.1 nmol/h/mg protein) was increased 11-fold compared to normal leukocytes (3.1 ± 0.5). K m values for IMP and NAD + of leukemic IMP dehydrogenase were 22.7 and 44.0 μM, respectively. XMP inhibited competitively with IMP and noncompetitively with NAD +. NADH exerted mixed type inhibition with respect to both IMP and NAD +. The inhibitory pattern of TAD was quite similar to that of NADH; however, the affinity of TAD to leukemic IMP dehydrogenase ( K i = 0.1 μM) was three orders of magnitude higher than the natural product NADH ( K i = 150 μM). These results contribute to an understanding of the mechanism of action of tiazofurin in the treatment of leukemia.

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells.

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.

177 TWO DISTINCT TARGET SITES ON IMP DEHYDROGENASE IN CHEMOTHERAPY

The inhibitory mechanisms of ribavirin 5′-monophosphate, (RMP), SM-108 nucleotide (bredinin 5′-monophosphate, BMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites, ribavirin, SM-108 and tiazofurin, were studied in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. RMP and BMP as well as XMP inhibited competitively with IMP and noncompetitively with NAD+. The inhibition by TAD was similar to that of NADU: uncompetitive with IMP and mixed type with NAD+. Ki values for RMP (0.8 uM), BMP (0.11 uM) and TAD (0.13 uM) were markedly lower than those for XMP (136 uM) and NADH (210 uM), and also the Km for IMP (23 uM) and NAD (65 uM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural metabolites, RMP or BMP with IMP-XMP and TAD with NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from inactivation by IMP or XMP but not by NAD or NADH. Ribavirin and tiazofurin together provided synergistic killing of hepatoma cells in clonogenic assays and the metabolic flux of de novo guanylate synthesis was also synergistically inhibited. The results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin against the 2 different ligand sites of IMP dehydrogenase. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)