Research Focus

Cancer is most commonly caused by the development of aberrant cell signaling pathways.  Modern pharmaceutical research thus seeks a clear understanding of these pathways in efforts to treat this all too prevalent illness.  A major focus in our laboratory are the mitogen-activated-protein-kinases (MAPKs).  MAPKs regulates cellular processes with remarkable efficiency and specificity.  They are mediators of numerous cellular signals, and are believed to play major roles in tumor formation and progression to metastasis.  One mitogenic pathway important in the pathogenesis of human cancer contains the Ras–>Raf–>MEK–>ERK module.  

    The role of ERK1/2 in cancer–The therapeutic potential of targeting the Ras pathway has been recognized for some time because, as noted by Robert Weinberg in The Biology of Cancer, it is probably the single most important mitogenic pathway in human cells, contributing to more than 30% of all known cancers.  A recent study identified mutant RAS and BRAF (BRAF statistics underlined) in cancer cell lines of various origins, including pancreas (100%), colon (35%, 18%), lung (18%, 3%), ovarian (15%, 4%), bladder (20%), neuroblastomas (11%) breast (2%), melanomas (9%, 59%), Glioma (11%), leukemias/lymphomas (17%), liver, and kidney (40).  The same study shows that BRAF oncogenic mutations occur in approximately 8% of human cancers (being particularly common in melanoma, colon cancer and non small lung carcinoma), with 80% of the somatic mutations corresponding to a single point mutation, V599E, which produces a perpetually active form of B-raf. 

Protein kinase biochemistry

Basis for substrate specificity; mechanisms of regulation and inhibition

A fundamental biochemical understanding of an enzyme, such as a protein kinase, provides a foundation from which to understand its cellular function.  We use recombinant DNA technology to engineer and express wild type and mutant forms of protein kinases in bacteria, or other host systems.  Using this approach we generally obtain milligram amounts of each kinase, which is purified using a series of affinity chromatography and FPLC protocols.  Probably the most pressing biochemical questions concerning protein kinases are related to the mechanisms by which they recognize cellular protein substrates and  the mechanisms by which they are regulated in cells.   Understanding these processes enables us to develop models for their cellular function as well as potential new strategies for inhibiting their cellular activity. 

When addressing these issues we have a host of cutting edge biochemical techniques to choose from.  Generally, these provide us with the means to determine kinetic and thermodynamic parameters associated with ligand binding events, conformational changes or chemical processes.  These studies are enriched through collaborations with structural laboratories.  One goal is to use high field NMR spectrometry to obtain snapshots of protein kinases binding protein substrates.  These studies can provide critical information on  the allosteric nature of protein kinase-substrate complexes as well as provide insight into the nature of substrate recognition.   Another goal is to determine structures of inhibitor–protein kinases complexes.  Often when inhibitors bind protein kinases the protein kinase undergoes a conformational change.  Knowledge of the conformationally altered structure provides the means to develop medicinal chemistry libraries of potential inhibitors as well as well as an opportunity to use computer-aided drug design.

Drug Discovery

The pharmaceutical industry is believed to spend approximately 30% of its research budget on the elucidation of protein kinase inhibitors, because some 200 protein kinases have been shown to have ties with human diseases. 

   Screening small-molecule libraries: through our close ties with the Texas Institute for Drug and Diagnostic Development (TI3D) we are very fortunate to have state-of-the-art high throughput automation facilities in Austin.  We have begun to utilize these facilities to screen small-molecule libraries for inhibitors of various protein kinases.   The goal of these screening initiatives is ultimately to identify novel leads from which potent inhibitors can be developed.

   

Modified Peptide Libraries: selective and potent Targeting Molecule inhibitors of interactions between protein kinases and their ligands are acquired by appending carboxylic acid containing compounds (CACCs) to naturally occurring consensus binding sequences, thereby greatly expanding their structural diversity and increasing their potential for potency.  The mechanism of action and specificity of these TMs are determined using enzyme kinetics, X-ray crystallography and the NMR methods, to facilitate further inhibitor development.

Cellular Enzymology

The profiling of protein kinase activities within cells  is one of our most exciting current challenges.  We wish to define when and where in a cell a protein kinase is active with reference to specific cellular events, such as the activation of a cell by a growth factor, or during the cell cycle.  Furthermore, we want to delineate the precise roles of protein kinases within the context of when and where they function.  To achieve these goals we are using our knowledge of chemistry to synthesize Targeting Molecules and fluorescent sensors of protein kinase activities, which can be switched on following delivery into cells.

Summary

Students with an interest in integrating the disciplines of chemistry and biochemistry, in the search of treatments for human disease, are strongly encouraged to apply.