PART IV. BIOCHEMISTRY AND EVALUATION OF ANTI-HIV THERAPEUTICS
Metabolism in Human Leukocytes of Anti-HIV Dideoxypurine Nucleosides" ARNOLD FRIDLAND,~ MARK A. JOHN SON,^ DAVID A. COONEY,' G . AHLUWALIA,' VICTOR E. MARQUEZ,' JOHN S. DRISCOLL,' A N D DAVID G . JOHNS bDepartment of Biochemical and Clinical Pharmacology St. Jude Children's Research Hospital Memphis, Tennessee 38101 'Developmental Therapeutics Program Division of Cancer Treatment National Cancer Institute National Institutes of Health Bethesda, Maryland 20892
INTRODUCTION A number of 2',3'-dideoxynucleosides inhibit the in vitro infectivity of human immunodeficiency virus (HIV), the etiologic agent of the acquired immunodeficiency syndrome (AIDS). Of the dideoxynucleosides studied to date the dideoxypurine nucleoside 2,3'-dideoxyadenosine (ddA) and its deaminated derivative 2', 3'-dideoxyinosine (ddI) exhibit particularly favorable therapeutic ratios and appear to be equally effective in the ATH-8 cell Phase I studies also have shown that ddI produces objective responses in patients with AIDS and severe AIDS-related ~ o m p l e x . ~ The antiviral activity of these drugs is thought to be mediated by inhibition of reverse transcriptase (viral RNA-directed D N A polymerase) in virus-infected ce1ls.l Hence phosphorylation of the nucleoside prodrug is required for activation to the putative active triphosphate. In earlier ~ t u d i e s ,we ~ . ~showed that ddA could be metabolized in human lymphoid cells to its metabolite ddATP but also was rapidly converted through the ubiquitous enzyme adenosine deaminase to ddI, which was cleaved to the purine base hypoxanthine by purine nucleoside phosphorylase. Additionally, attempts were made to see if prevention of deamination of ddA by the potent inhibitor 2'deoxycoformycin (dCF) could enhance the antiviral activity of the drug; however, this inhibitor did not greatly improve either the antiviral activity of ddA or its activation '8'
'This work was generously supported in part by PHS Grants 1 RO1 A127652 and 1 R01 CA43296; by (CORE) Grant P30 CA21765 from the National Institutes of Health; and by the American Lebanese Syrian Associated Charities. 205
206
ANNALS NEW YORK ACADEMY OF SCIENCES
to the active metabolite ddATP. These results suggested that there might be alternate modes by which dideoxypurine nucleosides could be metabolized to the putative active inhibitor of HIV replication in human cells. In this report, we would like to review our recent studies that deal with the enzymatic basis for, and regulation of, dideoxynucleotide accumulation from ddA, ddI, and a novel dideoxypurine nucleoside 2’,3’-dideoxy-2’-fluoroarabinosyladenine(2‘-Fdd-ara-A) (FIG. 1).
EXPERIMENTAL PROCEDURES Material Cells Human T lymphoid cells CCRF-CEM and Molt4 were maintained as described previously.6 The CAR-1, Py9, and CARA mutants, defined by their deficiencies in deoxycytidine (dCyd) kinase, adenosine (Ado) kinase, and both dCyd kinase and Ado kinase, respectively, were grown as described.6
Radiochemicals [2’,3‘-3H]ddAdo,30 Ci/mmol; [2,8J HIddAdo and [ V H ] carbocyclic 2’,3’-didehydro-2’,3’-dideoxyguanosine (Carbovir), both 1.3 Ci/mmol; [8-’H]ddguanosine (Guo), 2-3.5 Ci/mmol; [8-3H]dGuo, 16 Ci/mmol; and [2-3H]inosine(Ino), 35 Ci/mmol were obtained from Moravek Biochemicals (Bra, CA). [VHIdAdo, 20 Ci/mmol was from ICN Radiochemicals, Irvine, CA. [2,pr,3’-3H]ddIno was prepared by means of enzymatic deamination of either the sugar-labeled or base-labeled ddAdo using calf intestinal adenosine deaminase (Sigma Chemical Co., St. Louis, MO). 2’-F-[3H]-dd-ara-A(25 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO).
Chemicals ddADP and ddATP were purchased from Pharmacia (Piscataway, NJ), 2’-F-ddaraAMP was synthesized by means of the general method of Yoshikawa et al.,’ and 2‘-Fdd-ara-ATP was synthesized according to the general method of Kovacs and Otvos.8 ddAdo (NSC98700) and 2’-F-dd-ara-A (NSC613792) were obtained from the Drug Synthesis and Chemistry Branch (NCI, NIH). Carbovir was provided by the courtesy of Dr. Charles Litterst of the Developmental Therapeutics Branch (NIAID). All other nucleoside and nucleotide standards were purchased from Sigma Chemical Co. (St.
FRIDLAND et 01.: DIDEOXYPURINE NUCLEOSIDES
207
Louis, MO).’ Enzymes: adenosine deaminase (calf intestine) (200 U/mg) and alkaline phosphatase (E. coli; 45 U/mg) were purchased from Boehringer-Mannheim Biochemicals, Indianapolis, I N and Sigma Chemical Co., MO., respectively. PEI-cellulose TLC plates (20 x 20 cm) with UV,,, fluor were from Brinkman Instruments (Westburg, NY). Dithiothreitol and ATP were from Research Organic, Cleveland, OH.
Methods Metabolism Studies For metabolism studies, cells (Molt-4, CEM, or ATH-8) or CEM variants deficient in either dCyd kinase, Ado kinase, or in both enzyme activities growing in log phase were incubated at a density of about 1 X lo6 cells with either [3H]ddAdo, [3H]ddIno, or 2’-F-[3H]-dd-ara-A (10 p M final concentration, with 5 pCi/mL cell suspension), in the presence or absence of 5 p M dCF for 15 min prior to addition of the dideoxynucleo-
FIGURE 1. Structure of denine.
sides. After 5 h of incubation, cells were centrifuged and pellets were washed with cold phosphate-buffered saline and extracted with 60% aqueous methanol (- 20°C). After centrifugation, 200 p L of the supernatant was subjected to chromatography on an anion exchange Partisil 10 SAX column. One minute fractions were collected and radioactivity determined by liquid scintillation counting. Radioactive dideoxynucleotides in the HPLC fractions were identified by comparing their retention times with those of authentic standards.
Enzyme Assays Ado kinase and dCyd kinase were purified from CCRF-CEM cells or from human leukemic blasts obtained by leukopheresis from patients with leukemias, as de~cribed.~ The kinase activities were separated from nucleotidase activity and other nucleoside
208
ANNALS NEW YORK ACADEMY OF SCIENCES
phosphorylating enzymes. The standard reaction mixture for the assay of dideoxynucleoside phosphorylation contained 10 to 25 pg protein for dideoxynucleosides and 0.1 to 2 pg protein for natural substrates in a total volume of 26 p L containing 10 mM sodium HEPES buffer, pH 7.5; 3 mM dithiothreitol; 5 mM ascorbate; 20 mM MgCl,; 10 mM ATP; 15 mM phosphoenolpyruvate; 0.3 units pyruvate kinase; and 20 mM NaF. The assays were started by addition of substrate with incubation for 30 min at 37°C; the rate of phosphorylation was constant over the time period used. Reactions were terminated by addition of 75 p L of 1 mM unlabeled substrate in water. Aliquots (45 pL) were pipetted onto DEAE cellulose discs (2.4 cm Whatman DE8 1); the discs were dried at 75"C, washed three times with water, and counted. Background controls (zero time) were determined as above and subtracted from each test assay. Nucleoside phosphorylation by S'-nucleotidase was done with 2.4 mM 'H-labeled nucleoside (0.5 pCi) in a volume of 15 pL buffer containing 100 mM HEPES pH 7.4, 50 mM MgCl,, 500 mM KCl, 5 mM IMP, 3 mM dithiotreitol, 5 mM ATP, and 15 pg protein from the fast-flow Q/Blue Sepharose-purified nucleotidase(s). After 30 min at 37"C, reactions were terminated, 20-25% hydrolysis of the substrate IMP occurred during such incubations. Products from these reactions were measured by using polyethyleneimine (PE1)-cellulose thin layer chromatography. The thin layer plates were prespotted with carriers (10 nmol) and developed in water. Products and substrates were located visually by UV absorption, cut out, and quantitated by liquid scintillation counting as described.
Purification of Human Leukemic Cells 5'-Nucleotidase The cytoplasmic 5'-nucleotidase was purified from fresh leukemic blasts obtained by leukopheresis from patients. Briefly, a suspension of white cells freed of erythrocytes was homogenized in Tris HCl buffer, pH 7.4, containing 3 mM dithiothreitol, 10% (v/ v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM 0-phenanthroline, 5 mM benzamidine, and 0.5 mg soybean trypsin inhibitor. After centrifugation at 105,OOO g, the supernatant was stirred with 200 mL ion exchange resin (fast-flow Q-Sepharose that had previously been equilibrated with homogenization buffer). 5'Nucleotidase activity was eluted from the Q-Sepharose using a linear gradient buffer of 0-0.6 M KCl in homogenation buffer after packing the resin in a 2.5 x 30 cm column (Pharmacia XK-26). 5'-Nucleotidase activity was then further purified on a Blue Sepharose affinity gel (Reactive Blue 2 Sepharose CLdB, Sigma), which previously had equilibrated with 50 mM Tris buffer, pH 7.5, containing 3 mM dithiothreitol and 10% glycerol. The final 5'-nucleotidase activity was free of the major cellular nucleoside kinase activities and non-specific alkaline or acid-phosphatase and was stable, stored at -70°C for several months.
EXPERIMENTAL RESULTS AND DISCUSSION Dideoxynucleotide Phosphorylating Activities In extracts of human T lymphoid cells (CCRF-CEM) there are two enzyme activities, dCyd kinase and Ado kinase, that can phosphorylate ddA. As shown in TABLE
FRIDLAND et al.: DIDEOXYPURINE NUCLEOSIDES
209
1, both enzymes bind ddA with approximately the same affinity as their natural substrate 2'-deoxyadenosine (dAdo). The reaction velocity, however, is substantially slower with ddA than with dAdo, such that the maximum efficiencies are only 1 and 0.06%, respectively, of that reached with dAdo. Even at a concentration of 5 mM, no phosphorylation of ddI was detected with either dCyd kinase or Ado kinase. The results of preliminary experiments suggested that the phosphorylation of ddI might be catalyzed in the reverse reaction of a cytoplasmic 5'-nucleotidase. TABLE2 summarizes the procedure used to isolate this activity from CEM cells; a 24-fold purification of this enzyme was achieved. This enzyme preparation was free of detectable ATP-dependent nucleoside kinases and nonspecific phosphatases (with phenyl phosphate or ribose-5-phosphate as donors), was Mg2+-dependent, and exhibited an apparent K, of 2 mM MgC1, with 1 mM IMP (results not shown). The enzyme displayed a fairly sharp pH optimum at pH 7.4 (results not shown). 2, this partially purified 5'-nucleotidase activity from CEM As shown in FIGURE cells, in the presence of IMP as a phosphate donor, 50 mM MgCl,, 5 0 0 mM KCI, and
TABLE 1. Kinetics of Dideoxynucleoside Phosphorylation by Deoxycytidine Kinase
and Adenosine Kinasea Enzyme
Substrate
Km PM
dCyd kinase
dAdo ddA ddI dAdo ddA ddI
190 290 NDb 340 140 ND
Ado kinase
Vmax nmol/h/mg 340
Relative Efficiency V/K, 100
4
1.2
Metabolism in Human Leukocytes of Anti-HIV Dideoxypurine Nucleosides" ARNOLD FRIDLAND,~ MARK A. JOHN SON,^ DAVID A. COONEY,' G . AHLUWALIA,' VICTOR E. MARQUEZ,' JOHN S. DRISCOLL,' A N D DAVID G . JOHNS bDepartment of Biochemical and Clinical Pharmacology St. Jude Children's Research Hospital Memphis, Tennessee 38101 'Developmental Therapeutics Program Division of Cancer Treatment National Cancer Institute National Institutes of Health Bethesda, Maryland 20892
INTRODUCTION A number of 2',3'-dideoxynucleosides inhibit the in vitro infectivity of human immunodeficiency virus (HIV), the etiologic agent of the acquired immunodeficiency syndrome (AIDS). Of the dideoxynucleosides studied to date the dideoxypurine nucleoside 2,3'-dideoxyadenosine (ddA) and its deaminated derivative 2', 3'-dideoxyinosine (ddI) exhibit particularly favorable therapeutic ratios and appear to be equally effective in the ATH-8 cell Phase I studies also have shown that ddI produces objective responses in patients with AIDS and severe AIDS-related ~ o m p l e x . ~ The antiviral activity of these drugs is thought to be mediated by inhibition of reverse transcriptase (viral RNA-directed D N A polymerase) in virus-infected ce1ls.l Hence phosphorylation of the nucleoside prodrug is required for activation to the putative active triphosphate. In earlier ~ t u d i e s ,we ~ . ~showed that ddA could be metabolized in human lymphoid cells to its metabolite ddATP but also was rapidly converted through the ubiquitous enzyme adenosine deaminase to ddI, which was cleaved to the purine base hypoxanthine by purine nucleoside phosphorylase. Additionally, attempts were made to see if prevention of deamination of ddA by the potent inhibitor 2'deoxycoformycin (dCF) could enhance the antiviral activity of the drug; however, this inhibitor did not greatly improve either the antiviral activity of ddA or its activation '8'
'This work was generously supported in part by PHS Grants 1 RO1 A127652 and 1 R01 CA43296; by (CORE) Grant P30 CA21765 from the National Institutes of Health; and by the American Lebanese Syrian Associated Charities. 205
206
ANNALS NEW YORK ACADEMY OF SCIENCES
to the active metabolite ddATP. These results suggested that there might be alternate modes by which dideoxypurine nucleosides could be metabolized to the putative active inhibitor of HIV replication in human cells. In this report, we would like to review our recent studies that deal with the enzymatic basis for, and regulation of, dideoxynucleotide accumulation from ddA, ddI, and a novel dideoxypurine nucleoside 2’,3’-dideoxy-2’-fluoroarabinosyladenine(2‘-Fdd-ara-A) (FIG. 1).
EXPERIMENTAL PROCEDURES Material Cells Human T lymphoid cells CCRF-CEM and Molt4 were maintained as described previously.6 The CAR-1, Py9, and CARA mutants, defined by their deficiencies in deoxycytidine (dCyd) kinase, adenosine (Ado) kinase, and both dCyd kinase and Ado kinase, respectively, were grown as described.6
Radiochemicals [2’,3‘-3H]ddAdo,30 Ci/mmol; [2,8J HIddAdo and [ V H ] carbocyclic 2’,3’-didehydro-2’,3’-dideoxyguanosine (Carbovir), both 1.3 Ci/mmol; [8-’H]ddguanosine (Guo), 2-3.5 Ci/mmol; [8-3H]dGuo, 16 Ci/mmol; and [2-3H]inosine(Ino), 35 Ci/mmol were obtained from Moravek Biochemicals (Bra, CA). [VHIdAdo, 20 Ci/mmol was from ICN Radiochemicals, Irvine, CA. [2,pr,3’-3H]ddIno was prepared by means of enzymatic deamination of either the sugar-labeled or base-labeled ddAdo using calf intestinal adenosine deaminase (Sigma Chemical Co., St. Louis, MO). 2’-F-[3H]-dd-ara-A(25 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO).
Chemicals ddADP and ddATP were purchased from Pharmacia (Piscataway, NJ), 2’-F-ddaraAMP was synthesized by means of the general method of Yoshikawa et al.,’ and 2‘-Fdd-ara-ATP was synthesized according to the general method of Kovacs and Otvos.8 ddAdo (NSC98700) and 2’-F-dd-ara-A (NSC613792) were obtained from the Drug Synthesis and Chemistry Branch (NCI, NIH). Carbovir was provided by the courtesy of Dr. Charles Litterst of the Developmental Therapeutics Branch (NIAID). All other nucleoside and nucleotide standards were purchased from Sigma Chemical Co. (St.
FRIDLAND et 01.: DIDEOXYPURINE NUCLEOSIDES
207
Louis, MO).’ Enzymes: adenosine deaminase (calf intestine) (200 U/mg) and alkaline phosphatase (E. coli; 45 U/mg) were purchased from Boehringer-Mannheim Biochemicals, Indianapolis, I N and Sigma Chemical Co., MO., respectively. PEI-cellulose TLC plates (20 x 20 cm) with UV,,, fluor were from Brinkman Instruments (Westburg, NY). Dithiothreitol and ATP were from Research Organic, Cleveland, OH.
Methods Metabolism Studies For metabolism studies, cells (Molt-4, CEM, or ATH-8) or CEM variants deficient in either dCyd kinase, Ado kinase, or in both enzyme activities growing in log phase were incubated at a density of about 1 X lo6 cells with either [3H]ddAdo, [3H]ddIno, or 2’-F-[3H]-dd-ara-A (10 p M final concentration, with 5 pCi/mL cell suspension), in the presence or absence of 5 p M dCF for 15 min prior to addition of the dideoxynucleo-
FIGURE 1. Structure of denine.
sides. After 5 h of incubation, cells were centrifuged and pellets were washed with cold phosphate-buffered saline and extracted with 60% aqueous methanol (- 20°C). After centrifugation, 200 p L of the supernatant was subjected to chromatography on an anion exchange Partisil 10 SAX column. One minute fractions were collected and radioactivity determined by liquid scintillation counting. Radioactive dideoxynucleotides in the HPLC fractions were identified by comparing their retention times with those of authentic standards.
Enzyme Assays Ado kinase and dCyd kinase were purified from CCRF-CEM cells or from human leukemic blasts obtained by leukopheresis from patients with leukemias, as de~cribed.~ The kinase activities were separated from nucleotidase activity and other nucleoside
208
ANNALS NEW YORK ACADEMY OF SCIENCES
phosphorylating enzymes. The standard reaction mixture for the assay of dideoxynucleoside phosphorylation contained 10 to 25 pg protein for dideoxynucleosides and 0.1 to 2 pg protein for natural substrates in a total volume of 26 p L containing 10 mM sodium HEPES buffer, pH 7.5; 3 mM dithiothreitol; 5 mM ascorbate; 20 mM MgCl,; 10 mM ATP; 15 mM phosphoenolpyruvate; 0.3 units pyruvate kinase; and 20 mM NaF. The assays were started by addition of substrate with incubation for 30 min at 37°C; the rate of phosphorylation was constant over the time period used. Reactions were terminated by addition of 75 p L of 1 mM unlabeled substrate in water. Aliquots (45 pL) were pipetted onto DEAE cellulose discs (2.4 cm Whatman DE8 1); the discs were dried at 75"C, washed three times with water, and counted. Background controls (zero time) were determined as above and subtracted from each test assay. Nucleoside phosphorylation by S'-nucleotidase was done with 2.4 mM 'H-labeled nucleoside (0.5 pCi) in a volume of 15 pL buffer containing 100 mM HEPES pH 7.4, 50 mM MgCl,, 500 mM KCl, 5 mM IMP, 3 mM dithiotreitol, 5 mM ATP, and 15 pg protein from the fast-flow Q/Blue Sepharose-purified nucleotidase(s). After 30 min at 37"C, reactions were terminated, 20-25% hydrolysis of the substrate IMP occurred during such incubations. Products from these reactions were measured by using polyethyleneimine (PE1)-cellulose thin layer chromatography. The thin layer plates were prespotted with carriers (10 nmol) and developed in water. Products and substrates were located visually by UV absorption, cut out, and quantitated by liquid scintillation counting as described.
Purification of Human Leukemic Cells 5'-Nucleotidase The cytoplasmic 5'-nucleotidase was purified from fresh leukemic blasts obtained by leukopheresis from patients. Briefly, a suspension of white cells freed of erythrocytes was homogenized in Tris HCl buffer, pH 7.4, containing 3 mM dithiothreitol, 10% (v/ v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM 0-phenanthroline, 5 mM benzamidine, and 0.5 mg soybean trypsin inhibitor. After centrifugation at 105,OOO g, the supernatant was stirred with 200 mL ion exchange resin (fast-flow Q-Sepharose that had previously been equilibrated with homogenization buffer). 5'Nucleotidase activity was eluted from the Q-Sepharose using a linear gradient buffer of 0-0.6 M KCl in homogenation buffer after packing the resin in a 2.5 x 30 cm column (Pharmacia XK-26). 5'-Nucleotidase activity was then further purified on a Blue Sepharose affinity gel (Reactive Blue 2 Sepharose CLdB, Sigma), which previously had equilibrated with 50 mM Tris buffer, pH 7.5, containing 3 mM dithiothreitol and 10% glycerol. The final 5'-nucleotidase activity was free of the major cellular nucleoside kinase activities and non-specific alkaline or acid-phosphatase and was stable, stored at -70°C for several months.
EXPERIMENTAL RESULTS AND DISCUSSION Dideoxynucleotide Phosphorylating Activities In extracts of human T lymphoid cells (CCRF-CEM) there are two enzyme activities, dCyd kinase and Ado kinase, that can phosphorylate ddA. As shown in TABLE
FRIDLAND et al.: DIDEOXYPURINE NUCLEOSIDES
209
1, both enzymes bind ddA with approximately the same affinity as their natural substrate 2'-deoxyadenosine (dAdo). The reaction velocity, however, is substantially slower with ddA than with dAdo, such that the maximum efficiencies are only 1 and 0.06%, respectively, of that reached with dAdo. Even at a concentration of 5 mM, no phosphorylation of ddI was detected with either dCyd kinase or Ado kinase. The results of preliminary experiments suggested that the phosphorylation of ddI might be catalyzed in the reverse reaction of a cytoplasmic 5'-nucleotidase. TABLE2 summarizes the procedure used to isolate this activity from CEM cells; a 24-fold purification of this enzyme was achieved. This enzyme preparation was free of detectable ATP-dependent nucleoside kinases and nonspecific phosphatases (with phenyl phosphate or ribose-5-phosphate as donors), was Mg2+-dependent, and exhibited an apparent K, of 2 mM MgC1, with 1 mM IMP (results not shown). The enzyme displayed a fairly sharp pH optimum at pH 7.4 (results not shown). 2, this partially purified 5'-nucleotidase activity from CEM As shown in FIGURE cells, in the presence of IMP as a phosphate donor, 50 mM MgCl,, 5 0 0 mM KCI, and
TABLE 1. Kinetics of Dideoxynucleoside Phosphorylation by Deoxycytidine Kinase
and Adenosine Kinasea Enzyme
Substrate
Km PM
dCyd kinase
dAdo ddA ddI dAdo ddA ddI
190 290 NDb 340 140 ND
Ado kinase
Vmax nmol/h/mg 340
Relative Efficiency V/K, 100
4
1.2
