Biosynthesis of the Antiviral and Antifungal Pradimicin
Yi Tang, UC Los Angeles
Pradimicin is a glycosylated, polycyclic natural product that displays potent antiviral and antifungal activities. This proposal aims to understand the biosynthesis of pradimicin by ctinomadura hibisca and use the knowledge to rationally and combinatorially produce analogs of pradimicin that display enhanced activities and decreased hepatoxicity. Research performed in this proposal may lead to the development of novel antiviral/antifungal combination drugs that can be used to prevent and combat AIDS and AIDS induced fungal infections.
Recently, the FDA approved a third class of anti-HIV drugs known as entry and fusion inhibitors to block the earliest step of the viral replication cycle. Pradimicin inhibits the de novo infection of human T-cells with HIV-1 by specific binding to mannose residues of the HIV glycoprotein gp120. Infection of MT-4 human T-cell line with HIV was completely inhibited by treatment with 10 μg/mL of radimicin. In addition, pradimicins are fungicidal against a wide variety of fungi, including isolates that are resistant to other antifungal agents, such as amphotericin B. The exciting antiviral and antifungal activities of pradimicin led to a Phase I trial in the late 1990s. Unfortunately, despite the lack of toxicity in animal models, early phase I trials in human volunteers revealed hepatic toxicity. As a result, clinical investigation with pradimicin has been discontinued and analogs of pradimicin with improved pharmacological properties are needed.
The complexity of the pradimicin molecule has precluded efficient total synthesis and has severely impeded the development of novel pradimicin analogs. Engineered biosynthesis of pradimicin analogs thus represents an attractive and perhaps the only feasible option to accelerate the development of the next generation of pradimicin compounds exhibiting novel antiviral and antifungal properties. The goal of this UARP grant is to lay the foundation for engineered biosynthesis of pradimicin compounds by elucidating the metabolic pathway and reconstitute the pathway in a genetically amendable and highyielding heterologous host.
Three specific aims will be pursued during this proposal period:
- Sequencing and annotation of the pradimicin gene cluster. We will identify the biosynthetic gene cluster that encodes the enzymes involved in pradimicin assembly. We will sequence the gene cluster and annotate the cluster to reveal the genetic blueprint for pradimicin biosynthesis. We have obtained a cosmid-based genome library of A. hibisca and have identified four clones that may encode the pradimicin pathway.
- Reconstitution of aglycon biosynthesis in heterologous host. We will reconstitute the biosynthesis of the dihydrobenzo[α]napthacenequinone core in the heterologous host treptomyces coelicolor. Production of the aglycon in a genetically well characterized eterologous platform is important for engineered biosynthesis of pradimicin analogs. This is especially important since A. hibisca is genetically intractable and is difficult to culture. We have previously demonstrated successful reconstitution of the minimal pradimicin PKS in S. coelicolor (Lee and Tang, JACS, 2006). During this proposal period, we will expand on these findings and biosynthesize the complete aglycon core of pradimicin.
- Heterologous biosynthesis of the pradimicin and pradimicin analogs. We will reconstitute the aminoacylation and glycosylation steps that tailor the pradimicin molecule. These two downstream modification steps outfit the pradimicin molecule with its antiviral and antifungal activities. Understanding the tailoring steps will enable the systematic manipulation to the pradimicin scaffold and yield novel unnatural natural products.