7-Deaza-6-benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: Activities and selective toxicities
Introduction
The parasitic protozoan, Toxoplasma gondii, is the etiologic agent for toxoplasmosis, a parasitic disease wide spread among various warm-blooded animals including man [1]. Approximately a billion people worldwide, including 60% of the population in the US, are seropositive to T. gondii. Infection with T. gondii is asymptomatic (90% of cases) in the general population. By contrast, the disease represents a major health problem for immunocompromised individuals, such as AIDS patients, organ transplant recipient patients, and the unborn children of infected mothers [1], [2], [3], [4]. In such cases, toxoplasmic encephalitis is recognized as the most common cause of intracerebral mass lesions in AIDS patients and possibly the most commonly recognized opportunistic infection of the central nervous system [2], [3]. Congenital toxoplasmosis is as high as 1/1000 live births [3]. Effects range in severity from asymptomatic to stillbirth, with the most common ailments being retinochoroiditis, cerebral calcifications, psychomotor or mental retardation, and severe brain damage [3].
Despite these tragic implications, the current therapy has not changed in the past few decades. The efficacy of the current therapy for toxoplasmosis (a combination of pyrimethamine and sulfadiazine) is limited, primarily by serious host toxicity and ineffectiveness against tissue cysts. Furthermore, as many as 50% of patients do not respond to therapy. In addition, prolonged exposure to this regimen induces serious host toxicity such as bone marrow suppression and severe skin rashes forcing the discontinuation of the therapy [2], [3], [4], [5]. Other therapies, e.g., clindamycin, spiramycin or atovaquone, have met with limited success, particularly in the long-term management of these patients. Hence, there is a critical need to develop new and effective drugs with significant low host toxicity for the treatment and long-term management of toxoplasmosis.
Rational drug design is usually based on biochemical and physiological differences between the pathogen and the host. One potential target for chemotherapeutic intervention against T. gondii is purine metabolism. These parasites replicate rapidly and require large amounts of purines for the synthesis of their nucleic acids and other vital components. In contrast to their host, however, T. gondii are purine auxotrophs and must rely on the salvage of their purine requirements from the host [6], [7].
Another striking difference between toxoplasma and their host is the nature of adenosine salvage. Adenosine is preferentially incorporated into the parasite nucleotide pool by at least a 10-fold higher rate than any other purine nucleobase or nucleoside tested [8], [9]. Furthermore, adenosine is directly phosphorylated to AMP, from which all other purine nucleotides can be synthesized to fulfill the parasite purine requirements. This reaction is catalyzed by the enzyme adenosine kinase (EC.2.7.1.20) which is almost 10 times more active than any other purine salvage enzyme in this parasite [8]. This contrasts sharply with most mammalian cells where adenosine is predominantly deaminated by adenosine deaminase (EC 3.5.4.4) to inosine, which is then cleaved by purine nucleoside phosphorylase (EC 2.4.2.1) to hypoxanthine as previously reviewed [6], [7]. Neither of these two enzymes have any appreciable activity in T. gondii[8].
Structure–activity relationships [10], [11], [12], biochemical [11], [13], [14], metabolic [6], [13], [14], [15], [16], and molecular [17] investigations have demonstrated that the substrate specificity, as well as other characteristics of T. gondii adenosine kinase, differs significantly from those of the human enzyme, and have established the enzyme as an excellent potential chemotherapeutic target for the treatment of toxoplasmosis [6], [7]. It was also demonstrated that 6-benzylthioinosine, among other 6-substituted purine nucleoside analogues, is a substrate for the parasite, but not human adenosine kinase [6], [11], [13], [14], [15]. Furthermore, 6-benzylthioinosine was shown to be metabolized preferentially to the nucleotide level and becomes selectively toxic to T. gondii, but not their host, thereby acting as a subversive substrate [6], [11], [13], [14], [15]. Therefore, modification of the chemical structure of 6-benzylthioinosine could further potentiate its antitoxoplasmic efficacy.
Structure–activity relationships [10] also showed that 7-deazaadenosine is one of best ligands of T. gondii adenosine kinase. Therefore, we thought that elimination of the N7 of inosine would enhance the binding of 6-benzythioinosines to T. gondii adenosine kinase. Indeed, 7-deaza-6-benzylthioinosine analogues were better ligands than the parent compounds [12]. Herein, we report the testing of newly synthesized 7-deaza-6-benzylthioinosine analogues, with various substitutions at their phenyl ring [12], as subversive substrates for T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents in cell culture.
Section snippets
Chemicals
The 7-deaza-6-benzylthioinosine (1) analogues (1–24) were synthesized as previously described [12]. The chemical structures of these compounds are shown in Table 1. 7-Deazainosine and 6-benzylthioinosine were generously provided by Dr. Mohamed Nasr, Drug Development and Clinical Sciences Branch, NIAID. [5,6-13H]uracil was purchased from Moravek Biochemicals. RPMI-1640 medium from GIBCO BRL; penicillin G and streptomycin sulfate from Mediatech/Cellgro; fetal bovine serum (FBS) from HyClone
Evaluation of 6-benzylthioinosine analogues as alternative substrates for purified T. gondii adenosine kinase
7-Deaza-6-benzylthioinosine (1) and its analogues (Table 1) were tested as alternative substrates of T. gondii adenosine kinase. HPLC analysis of the substrates and products of the enzyme assays demonstrated that 7-deaza-6-benzylthioinosine (1) and its analogues were converted to their respective nucleoside 5′-monophosphates by T. gondii adenosine kinase. Fig. 1 shows the reversed-phase HPLC profile of the metabolism of one of these analogues, 7-deaza-6-(p-fluorobenzylthio)inosine (9), to its
Acknowledgments
This research was supported by the US Public Health Service Grant AI-52838 from the National Institute of Health. We thank Mudar Al Safarjalani, P.E. for computer programming and estimations of the IC50 values, and Marion Kirk for mass spectrometry analyses. The mass spectrometer was purchased by Grant S10RR13795 and UAB Health Services Foundation General Endowment Fund.
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