cAMP Antagonists of Protein Kinase as Breast Cancer Drugs

Institution: Scripps Research Institute
Investigator(s): Sanjay Adrian Saldanha, Ph.D. - Sanjay Adrian Saldanha, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0080 Award: $48,253
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

This is a collaboration with: 11FB-0080A -

Initial Award Abstract (2005)
Mounting evidence incriminates cAMP-dependent protein kinase (PKA) in breast cancer. However the ubiquitous nature of this kinase, and its requirement in normal cell processes, makes it unwise to non-specifically knock-out this enzyme. The PKA holoenzyme is composed of genetically distinct catalytic and regulatory subunits that form an inactive tetramer. Cell and animal models have established the Rlα regulatory subunit of PKA as a target for breast cancer drugs, and phase II cancer clinical trials are underway to determine the effectiveness of antisense therapy that prevents RIα synthesis.

In this project we will use an alternate approach to inactivate the breast cancer associated isoform of PKA (PKA-lα) by specifically targeting the Rlα subunit with a small molecule. A drug that prevents binding of cAMP to PKA-lα would prevent activation by this second messenger and thus lock this breast cancer associated isoform in to an inactive state. A recent x-ray crystal structure has revealed a novel conformation for the cAMP binding site on Rlα that can be exploited for drug discovery. Structure based design through virtual screening will be used to identify novel drug-like compounds that antagonize activation by cAMP.

The aims of this proposal will be to:

We plan to: (i) generate alternate conformations, through computer simulations, for the cAMP binding site on the A domain of Rlα that promote small molecule binding, (ii) employ “virtual ligand screening” against these structures to select a series of drug-like commercial compounds, (iii) test compounds nominated by virtual screenings in in-vitro and cell based assays, and (iv) determine the anti-tumor effects of identified ‘hits’ using breast cancer cell lines.

This research has as its ultimate goal the discovery of breast cancer drugs that act through novel mechanisms. The combination of structure-based drug discovery and assay development will serve as proof-of-concept for similar studies to be performed on novel breast cancer targets. It is envisioned that drugs emerging form this new approach will provide cheap and superior breast cancer small molecule drugs that act through new mechanisms and that overcome the drawbacks of existing treatments.


Final Report (2007)
Note: The PI changed institutions from Scripps to UCSD during the course of these studies.

Introduction One of the key proteins involved in cell signaling that is over-expressed in breast cancer is the RI? regulatory subunit of cAMP dependent Protein Kinase (PKA). RI? is a proven therapeutic target for breast cancer and antisense therapy is currently in clinical trials. Our hypothesis is that small molecules that selectively target PKA and either activate or inactivate this kinase may provide a novel therapy against breast cancer. A small molecule antagonist that locks type I? PKA-I? in an inactive state may act in the same way as antisense, but with the benefits in ease of administration (e.g. oral availability) offered by small molecules as drugs. Alternately, agonists of PKA may instead prove to be effective against breast cancer, since the natural molecule that activates PKA (cAMP) is known to promote apoptosis in breast cancer cells.

Progress towards specific aims
Several approaches were used in this research to identify novel and drug-like modulators of PKA. Initially, computational structure-based drug discovery was attempted. By using virtual ligand screening a series of a few hundred compounds predicted to bind with high affinity to the PKA holoenzyme protein complex was nominated for testing. However, these compounds showed no activity in assays with the purified PKA complex and this approach was abandoned. In developing the experimental techniques needed to test for PKA modulators a new assay was devised for the identification of PKA agonists or antagonists. This assay can be used to identify compounds that are selective for individual PKA isoforms and is compatible with high throughput techniques for screening large compound libraries. A series of drug-like small molecules and marine natural products was tested, but only analogs of cAMP were active towards PKA. In the last component of this fellowship the PI was given the opportunity to train at the Moores Cancer Center (UCSD) in cell-based drug screening techniques on a range of cancer cell lines. This cancer cell culture experience will prove useful in the PIs future research in assay development and oncology drug discovery.

Future directions
The results achieved during the 2 years of this fellowship have laid the groundwork for future studies to validate PKA as a drug target both for breast cancer as well as other diseases. Notably we have made collaborations with the NCI for them to screen their libraries with this new PKA assay. We are also collaborating in a medicinal chemistry driven approach to synthesize and test cAMP analogs with improved drug characteristics (i.e. cell permeability, stability and lack of toxicity in humans) and that are selective for individual PKA isoforms.

Impact
This fellowship has enabled the PI to perform drug discovery in academia, in collaboration with structural biologists, medicinal chemists, computational modelers and cancer biologists against a novel breast cancer target deemed to be of high-risk by the pharmaceutical sector.