Inhibition of avian myeloblastosis virus (AMV)reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction. The quinones were shownto function as electron acceptors as revealed by the catalytic oxidation of NADHby Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity.The kinetics of inhibition of AMVreverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5,8-dihydroquinoline-5,8-dione, 5,8-dihydroisoquinoline-5,8-dione and 1 ,4-naphthoquinone, were studied. These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule. In contrast, the quinones with bulky substituents, i.e. , 7-(2-nitrophenethylamino)-5,8-dihydroisoquinoline-5,8-dione and 7-methoxy-6-methyl-3-piperidino-5,8-dihydroisoquinoline-5,8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADHby C. kluyveri diaphorase. Based on these observations, the existence of a specific site of interaction on the enzymemolecule, referred to as a quinone pocket, was proposed. The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM). Access of SKMto a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group. This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidationreduction system as comparedwith those of STN. STNshowed specificity for AMVreverse transcriptase in comparison with calf thymus DNAdependent DNApolymerase (DDDP) alpha and Escherichia coli DDDPI3). By using various quinones, the importance of the role of the quinone group in the biological activities of STN and good correlation between electron acceptor activities in the oxidation of NADH by Clostridium kluyveri diaphorase and inhibitory activities against AMV reverse transcriptase were reported previously4~6). Depending on these observations, the specificity shown by STNare considered to derive from the
Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV)and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies.Against AMVreverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH,with the ID50 values ranging 2~8 jug/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but notshowed collateral sensitivity to both STN-COOHand STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.Streptonigrin (STN-COOH, 1) was first isolated as an antitumor antibiotic, especially active against osteosarcomas, from Streptomyces flocculus in 19601~3). However, the severe side effects of 1, mainly due to bone marrow depression, resulted in the discontinuation of its clinical application. It has been reported that STN-COOCH3 (2) is clinically superior to 1 as an antitumor agent in terms of chemotherapeutic coefficient4 > 5). Besides antitumor activity, the strong inhibition of avian myeloblastosis virus (AMV)and human immunodeficiency virus (HIV) reverse transcriptasesby STN-COOH(1) has been reported6»7). Since reverse transcription is a pivotal step in the replication of retroviruses, inhibitors of this enzyme have been a target of the search for chemotherapeutics against retrovirus-related diseases, including acquired immune deficiency syndrome (AIDS). The synthesis of novel hydrazides, a hydroxamic acid derivative, amides and amino acid derivatives of streptonigrin such as STN-CONH(CH2)3N(CH3)2 (12) and , and the comparison of their biological activities with those of 1 and STN-COOCH3 (2) have been described previously50. The inhibition of AMVreverse transcriptase by the individual hydrazides, amides and amino acid derivatives compared favorably to that of 1, while 2 was essentially devoid of this activity. In contrast, the prominent inhibition of the growth of murine lymphoblastoma L5178Y cells was not observed with the amides and amino acid derivatives except for 12 and STN-CONH(CH2)3NH(CH2)4NHCO-STN, suggesting the role of the Af-alkylamino group in the substituent.
Streptonigrinis a potent inhibitor of avian myeloblastosis virus (AMV)reverse transcriptase in a non-competitive manner by direct interaction with an enzyme molecule0. In addition to streptonigrin, inhibition of AMVreverse transcriptase was observed with another quinone antibiotic, sakyomicin A2). These observations prompted us to extend our search for inhibitors of AMVreverse transcriptase to various carbocyclic and heterocyclic quinones3~5). Several naphthoquinone, quinoline quinone and isoquinoline quinone derivatives were found to inhibit AMV reverse transcriptase to the same extent as streptonigrin. The inhibitory activities against AMV reverse transcriptase of these quinone compounds including both streptonigrin and sakyomicin A were well correlated with their catalytic activities in the oxidation of NADH by rat liver mitochondria or Clostridium kluyveri diaphorase, which were dependent on their potential to accept electrons from NADH catalyzed by diaphorase and autoxidation to quinones with the simultaneous generation of hydrogen peroxide by transferring electrons to molecular oxygen. 1,4-Benzoquinone was an exception to this general concept, however, showing inhibition of reverse transcriptase without any marked effect on the oxidation of NADHby C. kluyveri diaphorase in terms of generation of hydrogen perioxide. Contrary to the earlier proposition by Wick and Fitzgerald6) that the generation of semiquinone and/or oxygen radical triggered the re-actions resulting in the inactivation of reverse transcriptase, we demonstrated that the induction of semiquinone and accompanying superoxide anion had no effect on the inhibition of AMVreverse transcriptase by quinones including, in particular, sakyomicin A7).In the preceding papers3~5), we proposed that the naphthoquinone and quinoline quinone moieties were the minimumrequisites for the biological activities of sakyomicin A and streptonigrin, respectively, based on the biological properties of quinoline quinones, naphthoquinones and 1,4-benzoquinone. Interestingly, isoquinoline quinones were as potent as inhibitors of AMVreverse transcriptase as quinoline quinones. Furthermore, inhibition of AMV reverse transcriptase was not found to differ whether ortho-or /?
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