Hockensmith, Joel W.
Associate Professor, Biochemistry and Molecular Genetics
- BS, Pharmacy, Philadelphia College of Pharmacy and Science
- PhD, Biochemistry, University of Rochester
Novel antiprotozoan and anticancer compounds from antibiotic-resistant bacteria
Biochemistry and Molecular Genetics
Studies concerning the molecular interactions of proteins that bind to nucleic acids are being conducted to elucidate the structure-function relationships that occur during DNA metabolic processes (e.g. synthesis, repair, transcription). We are especially interested in how multiple protein complexes move along nucleic acids and recognize the particular DNA structures at which enzymatic activity occurs. In particular, we have recently focused on the regulation of DNA metabolic processes by DNA-dependent ATPases. These enzymes require DNA for ATP hydrolysis, which subsequently leads to alterations in DNA metabolic function. We have identified, purified, cloned, and overexpressed a DNA-dependent ATPase from eukaryotic tissues. Our studies have many phases, which include: enzymology of the ATPase, molecular biological manipulation of the coding sequence, regulation of enzyme activity, exploration of the role of the enzyme in the cell, and the development of novel inhibitors of enzymatic activity. We are using the inhibitors of DNA-dependent ATP hydrolysis to disrupt DNA metabolic processes, and are developing novel chemotherapeutic strategies for treatment of cancer and parasitic diseases (see below). Additional studies are aimed at elucidating structure-function relationships that occur during DNA metabolic processes. As an example, DNA replication requires that the replication protein complexes move along the DNA template on a millisecond time scale. Consequently, we have developed a method of examining protein-nucleic acid interactions on a sub-millisecond time scale using a Nd-YAG laser, which produces a 5 nanosecond pulse of ultraviolet light. Irradiation of a protein-nucleic acid complex produces a covalent bond between protein and nucleic acid, effectively freezing the protein-nucleic acid interaction for further evaluation by other methods. We use the laser cross-linking methodology in a number of different systems to ask a variety of questions about the geometry of a DNA-protein complex. For example: what is the geometry of such a complex? How does the geometry change in static versus synthesis modes? How does a replication complex find a 3'-hydroxyl primer-template junction? What is the mechanism of translocation along a DNA template? These questions lead to others that deal with the energy requirements for protein movement and how proteins find each other and their substrates.
A recent focus of our laboratory is intervention in SWI/SNF-dependent metabolic pathways for control of tumor cell growth. The SWI2/SNF2 family of DNA-dependent ATPases participates in a number of DNA metabolic processes, including DNA repair, chromatin remodeling, recombination and transcription. Individual members of this family of proteins have been demonstrated to interact with and regulate a limited set of transcription factors including steroid receptors (estrogen receptor, androgen receptor, etc.), p53, C-MYC, the retinoblastoma protein and BRCA1. Many of these factors have been investigated for their role in the development and/or progression of breast cancer or prostate cancer, although the steroid receptor has been the primary target for chemotherapy. We propose that an alternative target might be the underlying distinctive feature of the SWI2/SNF2 proteins, which is a molecular motor domain comprised of an adenosinetriphosphatase (ATPase) catalytic site functionally coupled to a DNA binding site. The latter site effects DNA-dependent ATP hydrolysis when occupied and the peptide elements comprising this domain play an important role in the allosteric modulation of ATP hydrolysis. We observe that a family of inhibitors called phosphoaminoglycosides can alter the function of this core. We have found them to inhibit the SWI2/SNF2 molecular motor, inhibit cell growth and exert selectivity with respect to prostate cancer cell type, which is consistent with the proposed role of SWI2/SNF2 family members regulating androgen receptor function. Regulation of the SWI2/SNF2 family members is a novel and important finding since these proteins regulate a limited number of transcription factors that are intimately involved with cell growth and differentiation. We are exploring the SWI2/SNF2 proteins as novel targets for therapeutic exploitation with specific interest in control of cell growth by the phosphoaminoglycosides.
The advent of drug-resistant strains of bacteria and protozoans is expected to exacerbate the already heavy toll parasitic diseases exact on human life and health. Development of new paradigms for controlling these parasites is important and may come through a basic understanding of DNA metabolic events. Our extensive biochemical analysis of a nuclear enzyme's interactions with DNA has led to the discovery of a mechanism to disrupt ATP-dependent DNA metabolic events. Antibiotic-resistant bacteria are the source of potent, naturally occurring inhibitors of eukaryotic DNA-dependent ATPase activity. These are the first reported inhibitors that are specific for DNA-dependent ATP hydrolysis. The DNA-dependent ATPase domain targeted by these inhibitors is found in proteins that play integral roles in many DNA metabolic processes; including DNA replication, transcription, repair, recombination and histone remodeling. Three inhibitors have been demonstrated to be toxic to protozoans (Plasmodium falciparum (Malaria), Entamoeba histolytica, Leishmania chagasi) but not bacteria. Five additional inhibitors have been prepared and demonstrated to be inhibitory for the nuclear enzyme in vitro. The eight derivatives range widely in potency (50-fold) and are generally more than a thousand-fold more potent than the parent antibiotic. The targeting of specific nuclear enzymes leading to disruption of the protozoan life cycle provides an exciting development in the treatment of protozoan diseases. We are attempting to expand and refine our understanding of DNA metabolic events in protozoans and to evaluate mechanisms that could lead to the death of protozoans. Our studies aim to facilitate the development of DNA-dependent ATPase inhibitor species specific for protozoan growth.