Paul C. Zamecnik

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A): Its role in cellular metabolism

Previous work showed that the concentration by Ap/sub 4/A increased 50- to 100-fold when mammalian cells in tissue culture passed from the growth-arrested or resting phase of their cell cycle into the early S phase, prior to cell division. In pursuit of this observation, it was then found that addition of Ap/sub 4/A to permeabilized, resting tissue culture cells promptly induced the formation of multiple small replication eyes and restored DNA synthesis. A relationship of a unique nucleotide, Ap/sub 4/A, formed in the back-reaction of the first step in protein synthesis, to the initiation of DNA synthesis was uncovered. The role of Ap/sub 4/A as a signal molecule linking the first event in protein synthesis to the initiation of DNA synthesis is currently the subject of much further investigation. The possible role of Ap/sub 4/A as a trigger for continued DNA synthesis in the relentless growth of cancer tissue is examined.

Diadenosine 5′,5‴-p1,p4 - tetraphosphate, a potential antithrombotic agent

Diadenosine tetraphosphate (AP4A), a competitive inhibitor of ADP-induced platelet aggregation, was tested as an antithrombotic agent in a rabbit intracarotid thrombosis model previously shown to be sensitive to antiplatelet agents. Eighty-four percent of control rabbits formed clots. The infusion of AP4A at a dose of 50 mg/kg over 2 hours reduced the incidence of thrombosis to 56% (p less than 0.05). Blood AP4A increased 125-fold at the end of infusion, but was completely cleared within 10 minutes. Plasma ATP showed bimodal early and late increases. Platelets recovered from AP4A-treated rabbits exhibited a pattern of reduced reactivity to ADP, but not to collagen, similar to platelets exposed to AP4A in vitro. This study shows that AP4A may be a potentially useful antithrombotic agent.

Synthesis and anti-HIV activity of oligoribonucleotides and their phosphorothioate analogs.

In previous reports, we demonstrated that replication of human immunodeficiency virus (HIV) could be inhibited by unmodified phosphodiester oligodeoxynucleotides complementary to HIV RNA.’x2 However, a relatively short half-life of unmodified oligodeoxynucleotides in serum and in cells, due to the presence of nucleases, limits their potential usefulness in vivo. To overcome this limitation, we and others have studied internucleotide phosphate backbone modified oligonucleotides, such as methyl phosphonate~,~.~ phosphor amid ate^,^ and pho~phorothioates.~-~ Several oligonucleotide conjugates such as oligonucleotide-cholesteryl conjugateslO and oligonucleotide-polylysine conjugates” have also been studied. These analogs of oligodeoxyribonucleotides are resistant to nucleases and have generally increased hydrophobicity, which provides longer survival time in vivo and may increase cell membrane permeability. Oligodeoxynucleotide phosphorothioates are effective in inhibiting HIV replication in tissue culture (ICso) at a concentration of approximately 1 x M.9 The anti-HIV activity of oligodeoxyribonucleotide phosphorothioate is found to be “relatively sequence independent” against freshly infected cells, but bear in mind that it is difficult for a computer to define a completely random 20-mer sequence against HIV and one that does not activate RNase H. There is, however, striking sequence-specific activity against HIV chronically infected cell^.^.^ The cholesteryl conjugates of oligodeoxyribonucleotide phosphorothioates were found to be more effective than their unconjugated counterparts, but cholesterol conjugation increases nonsequence-specific activity as well.]” The inhibition of HIV replication by an oligodeoxyribonucleotide phosphorothioate is length dependent, a 25 mer being more active than 15-20 mers.I2 An increase in length to 30 mer or 35 mer had little or no enhanced effect on anti-HIV activity over that of the 25 mer. Our continued interest in developing antisense oligodeoxynucleotides and their various analogs as anti-HIV agents has stimulated us to investigate oligoribonucleotides and their phosphorothioate analogs as anti-HIV agents.

Phosphoramidate, Phosphorothioate, and Methylphosphonate Analogs of Oligodeoxynucleotide : Inhibitors of Replication of Human Immunodeficiency Virus

Abstract Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The modification of oligomers include replacement of phophodiester backbone with phosphorothioate, methylphosphonate and various phosphoramidates. Cells infected for four days, then treated with the antisense oligomers also showed inhibition of viral expression.

History of Antisense Oligonucleotides

Biological science is a rapidly flowing experimental stream, at times encountering a dam that impedes further progress. At such a pomt, a single crack may induce a major breakthrough Discovery of the double helical structure of DNA in 1953 (1) caused such an event, with flooding of new information into the area now known as molecular biology.

5',5'''-P1, P4 diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication.

The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single-stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not interact with Ap4A this DNA polymerase was not a multiprotein form of polymerase alpha and the synchrony of the wheat germ embryos was not known. A possible tie between protein-synthesizing systems and the regulation of proteins involved in DNA replication may exist. The requirement of protein synthesis for the initiation of DNA replication has long been known. Also, it is well established that many temperature-sensitive mutants for tRNA synthetases are also DNA-synthesizing mutants. More investigation in this area may be warranted.(ABSTRACT TRUNCATED AT 400 WORDS)

Diagnosis of storage pool deficiency by determination of diadenosine 5', 5'''-P1,P4-tetraphosphate in whole blood.

Platelets from cat and cattle with Chediak-Higashi disease were found completely devoid of Ap4A as measured by high performance liquid chromatography. Using a very sensitive firefly biolumnescence method 6% of the normal content of Ap4A was, however, found in platelets from sick animals. A content of Ap A of 1.90 +/- 0.11 X 10 M (means +/- SEM, n = 10) was found in whole normal human blood as measured by firefly bioluminescense method in trichloroacetic acid extracts of the blood samples. This concentration corresponds to the contribution from the platelets, thus the contribution of Ap4A from erythrocytes and the buffy-coat is negligible. Using the same method an Ap4A contents in platelets of 0.063 and 0.021 nmol/mg of protein compared to the normal content of 0.42 nmol/mg of protein (1) was found in two patients with severe myeloproliferative disorder calculated in this way on basis of platelet counts and on the assumption that 10(11) platelets contain 189 mg of protein (2). Comparison of these figures with parallel HPLC analyses on acid extracts of platelets isolated from the same patient were in agreement. The storage pool deficiency of adenine nucleotides in this disease found by others on basis of release experiments (3) can thus be diagnosed by rapid and simple measurements of Ap4A in whole blood using the advantage of Ap4A being a specific components stored in dense granules.

Solid phase synthesis of oligonucleotides carrying puromycin at 3′-terminal

Abstract For incorporation of puromycin at 3′-end of oligonucleotides or their analogs, suitably protected puromycin has been attached to controlled pore glass (CPG).

Use of Drugs Targeted to Inhibit Different Stages of the HIV Life Cycle in the Treatment of AIDS

Acquired immune deficiency syndrome (AIDS) is a global disease which has presented extraordinary challenge to the scientific community in terms of understanding the pathogenesis, treatment and prevention of the disease. With the identification of human immunodeficiency virus (HIV) as the etiological agent (1–3), it is now possible to investigate approaches to develop treatments for the disease and vaccines for the prevention of the disease. With an estimated six to ten million HIV-1 infected individuals worldwide and over 160,000 known cases of AIDS in the United States, it is extremely important and urgent to look for strategies to control the disease by antiviral therapy.