John A. Montgomery

Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Abstract The unnatural diastereoisomer of l -5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [ 3 H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K i , were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.

Broad-spectrum antiviral activity of the carbocyclic analog of 3-deazaadenosine

The carbocyclic analog of 3-deazaadenosine (C-c3 Ado) was found to inhibit in vitro the replication of several DNA and RNA viruses, including vaccinia, reo, measles, parainfluenza and vesicular stomatitis, at a concentration of 0.2-1 microgram/ml, while not being toxic for the host (primary rabbit kidney, HeLa, Vero) cells at a concentration of 400 micrograms/Ml. In its activity against vesicular stomatitis virus, parainfluenza virus, measles and reo virus, C-c3 Ado proved about 100 times more potent than other established broad-spectrum antiviraL agents such as ribavirin (virazole) and (S)-DHPA ((S)-9-(2,3-dihydroxypropyl)adenine). In vivo, C-c3 Ado protected newborn mice against a lethal infection of vesicular stomatitis virus when administered as a single dose of 20, 100 or 500 micrograms per mouse 1 h after virus infection.

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

Abstract The synthesis of a new analog of adenosylhomocysteine (AdoHcy), 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, is described. The compound is a non-competitive inhibitor of AdoHcy hydrolase, with a K i of 0.4 mM, as compared to a K i of 3 μM for 3-deazaadenosine, a competitive inhibitor of the enzyme. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine is not hydrolyzed by AdoHcy hydrolase. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine when tested has a selective antiviral activity against Rous sarcoma virus in chick embryo cells, and against Gross murine leukemia virus in mouse embryo cells. Possible mechanisms of this anti-viral activity are discussed.

Activity of adenos1ne analogs against a cell culture, line resistant to 2-fluoroadenine☆

Abstract By serial transfer in the presence of 2-fluoroadenine, a subline of H.Ep. #2 cells in culture was selected for high resistance to 2-fluoroadenine. The resistance was stable when the cells were cultured in the absence of 2-fluoroadenine. The resistant cells were essentially devoid of AMP pyrophosphorylase activity, but retained IMP and GMP pyrophosphorylase and purine nucleoside kinase activities. Intact growing cells of the parent and resistant lines did not differ in capacity to incorporate into polynucleotides 14 C from adenosine-8- 14 C and hypoxanthine-8- 14 C, but, as compared to sensitive cells, the resistant cells incorporated 14 C from adenine-8- 14 C very poorly. The resistant cells were cross-resistant to adenine analogs but did not differ from the parent cells in sensitivity to a series of adenosine analogs. Many nucleosides were much more toxic to the parent cell line than were the corresponding free bases. These results suggest that a purine nucleoside kinase, presumably adenosine kinase, is the critical enzyme in the. activation of these cytotoxic analogs of adenosine.

Human thymidylate synthetase—III: Effects of methotrexate and folate analogs

Abstract The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N 10 ; and (2) tetrahydrofolate (H 4 PteGlu) analogs modified at N 5 and N 10 . With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its K i ; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its K i to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N 5 and N 10 or by substitution at the N 5 position were found to be poor inhibitors under our assay conditions.

Gene Therapy of Cancer: Activation of Nucleoside Prodrugs with E. coli Purine Nucleoside Phosphorylase

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

Comparison of the properties of metabolites of CCNU

Abstract Properties of the six isomeric N -(2-chloroethyl- N ′-(hydroxycyclohexyl- N -nitrosoureas, which have been identified by other investigators as metabolites of N -(2-chloroethyl- N ′-cyclohexyl- N -nitrosourea (CCNU), have been compared with those of CCNU and 2-[[[(2-chloroethyl)nitrosoamino]-carbonyl]amino]-2-deoxy- D -glucose (chlorozotocin). There are significant differences in the physicochemical, chemical, and biological properties of these metabolites, and the properties of some of them are significantly different from those of CCNU and chlorozotocin. The position of the hydroxy group and the steric configuration of the compound markedly affect the alkylating and carbamoylating activities of the compounds. The metabolites having the higher alkylating activities and the lower carbamoylating activities produce lethal toxicity to mice at lower molar doses but have somewhat better therapeutic indexes. The data are consistent with the hypothesis that the biological effects observed following the administration of CCNU are due to a large extent to the major metabolites with lesser effects contributed by the minor metabolites. Some of the metabolites have slightly better therapeutic indexes against murine leukemia L1210 than CCNU and chlorozotocin, and they are more soluble in water than CCNU but are active against both intraperitoneally implanted and intracerebrally implanted L1210 leukemia. There might be some therapeutic advantage to administering one of the minor metabolites instead of CCNU or chlorozotocin to cancer-bearing animals.

Preclinical Antitumor Activity of 2-Chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (C1-F-Ara-A)

Abstract Cl-F-ara-A, an analog of fludarabine, was evaluated against a spectrum of tumor systems in culture and in mice. The compound exhibited significant cytotoxicity against a variety of human tumor cell lines. More importantly, the compound showed selectivity in vivo, with excellent activity being demonstrated against human colon and renal tumors. Human nonsmall cell lung and prostate tumors were also sensitive in vivo to the compound, albeit at a reduced level.

Approaches to antiviral chemotherapy.

Most of the drugs used today in the treatment of viral infections in man are purine-pyrimidine antimetabolites that interfere with viral replication. Work at Southern Research Institute has identified a number of compounds of this type with promising antiviral activity in both cell culture and rodent test systems. By far the most active and selective agents are carbocyclic nucleoside analogs in which the oxygen of the furanose ring is replaced by a methylene group. The effects of this change on the metabolism and antiviral activity of these compounds is discussed below.