Research Overview
Biosynthesis of Aflatoxin
Aflatoxin is a potent environmental carcinogen produced by Aspergillus fungi that infect grains and foodstuffs. A Type I polyketide synthase (PKS) and a pair of specialized fatty acid synthase subunits (FAS-1 and FAS-2) form a complex (1.3 MDa!) to synthesize norsolorinic acid. These large polydomainal proteins constitute an important experimental system to study Type I enzymes and address the central question of “programming” by these proteins; that is, how do these iterative catalysts “know” what product to make? Then a sequence of about 15 redox reactions ensues, often mediated by cytochromes P-450. The broader superfamily of these oxygenases is well-known to play central roles in secondary metabolism, and they are seen to figure prominantly in the intermediate steps of aflatoxin biosynthesis to carry out inter alia two unusual skeletal cleavage and rearrangement reactions that are also under investigation [Minto and Townsend, Chem. Rev. 1997, 97, 2537-2555; Neely and Townsend, J. Org. Chem. 1999, 64, 4050-4059; Graybill et al., J. Amer. Chem. Soc. 1999, 121, 7729-7746].
DNA Cleavage by Calicheamicin
Calicheamicin (CLM) is a central member of the enediyne antitumor antibiotic family. In in vitro assays CLM is approximately 1000 times more potent than adriamycin. This extremely high activity is thought to derive from its unusual ability to cause double strand breaks in DNA to lead on to apoptosis at pmolar concentrations. We have learned a great deal about its reductive activation [Chatterjee et al., J. Am. Chem. Soc. 1995, 117, 8074-8082; 1996, 118, 1938-1948.] and DNA recognition properties [Kuduvalli et al., Biochemistry 1995, 34, 3899-3906]. These insights have been used to develop a general method to attach a non-sequence selective derivative of CLM to homeodomains to prepare affinity cleavage reagents useful for mapping the affinity sites of DNA binding proteins, interfering with transcription and delivering genotoxic drug molecules [Wu et al., J. Amer. Chem. Soc. 2000, 122, 12884-12885].
Biosynthesis of β-Lactam Antibiotics
The β-lactam antibiotics, exemplifed by the penicillins and cephalosporins, are mainstays in the treatment of infectious diseases in humans. Resistance, however, is growing to antibiotics and threatening established therapeutic regimens. Three classes of $beta;-lactam antibiotics are known apart from penicillin and cephalosporin. These are the inhibitor clavulanic acid, the carbapenems, the simplest of which is carbapen-3-em-carboxylic acid, and the monocyclic $beta;-lactams as the nocardicins and monobactams. These have important properties to combat resistant organisms and provide surprisingly divergent solutions to the biochemical problem of structural diversity and assembly of their highly strained ring systems. Mechanistically distinct biochemical pathways have been uncovered to each of these three structural types, often employing new reactions evolved in unexpected ways from known enzyme classes [Li et al., J. Amer. Chem. Soc. 2000, 122, 9296-9297; Khaleeli et al., J. Amer. Chem. Soc. 1999, 121, 9223-9224. (see ‘Perspectives’ article: Science 2000, 287, 818-819); Bachmann et al., Proc. Nat'l. Acad. Sci., U.S.A. 1998, 95, 9082-9086; Biochemistry 2000, 39, 11187-11193]
In connection with this problem, the recent X-ray structure of the adenylation domain shown on the homepage has led the Townsend lab to a powerful algorithm that is predictive of amino acid substrate activation by the broad class of non-ribosomal peptide synthetases (NRPSs). For both the penicillin/cephalosporin, and the monocyclic families of β-lactams, modular NRPS enzymes are involved in assembly of their biosynthetic precursors [Challis et al., Chemistry & Biology 2000, 7, 211-224]. To use this algorithm, click here.
Fatty Acid Synthase Inhibition
An extensive web of collaborative research has been developed over several years with scientists in the Johns Hopkins School of Medicine. The unifying theme of this effort has been the inhibition of fatty acid synthase as a therapeutic approach to the treatment of a variety of human cancers, infectious diseases caused by mycobacteria, for example, tuberculosis, and a potential breakthrough therapy for obesity.
Fatty acid synthase (FAS) is massively upregulated in many cancers of, for example, the breast, prostate, colon, ovary etc. In animal models these human cancers have been demonstrated to be selectively killed by FAS inhibitors such as the natural product cerulenin and by inhibitors designed and synthesized in the Townsend laboratory [Kuhajda et al., Proc. Nat’l. Acad. Sci., U.S.A. 2000, 97, 3450-3454; Pizer et al., Cancer Res. 2000, 60, 213-218]
