The objective of this proposal is to explore the effects of autophagy modulation in the context of cancer. Autophagy is a process in which the cell consumes its own components in response to metabolic stress. The phosphoinositide 3-kinases (PI3-Ks) are among the most frequently mutated proteins in cancer. Inhibition of the class I PI3-Ks has been suggested to induce autophagy. Furthermore, inhibition of PI3-Ks leads to a decrease in the uptake of glucose and amino acids. One hypothesis is that deficiency in nutrients can lead to an increase in the AMP/ATP ratio, subsequent activation of AMP-activated protein kinase (AMPK) and induction of autophagy. This may be a mechanism that prevents cancer cells that are treated with PI3-K inhibitors from undergoing apoptosis.
In Aim 1 of the proposal, a panel of small molecule PI3-K inhibitors will be used to dissect the autophagy response upon inhibition of the various PI3-K isoforms in cancer cells. The effects of autophagy modulation by small-molecule AMPK regulators will be explored in the context of PI3-K inhibition.
In Aim 2, new chemical genetic tools will be developed for the study of autophagy. AMPK, a master sensor of cellular energy, is emerging as a key enzyme in autophagy regulation. However, chemical modulators of AMPK have been lacking. For example, the only known AMPK inhibitor, compound C, is known to inhibit other kinases. In this aim, using site-directed mutagenesis and organic synthesis, an inhibitor-sensitive allele of AMPK will be developed such that selective inhibition of AMPK can be achieved among the kinase superfamily. Besides non-specific inhibition, until recently, AMPK activation has been achieved by indirectly altering the AMP:ATP ratio or by weak allosteric activation with a nonspecific AMP mimetic, AICAR. A family of thienopyridone AMPK activators has recently been developed, but use of these compounds has not been extensively validated. Therefore, a specific and potent chemical genetic AMPK-activator will be developed using random mutagenesis to sensitize AMPK to activation by a thienopyridone derivative that does not activate wild-type AMPK. Finally, in Aim 3, uncoordinated 51-like kinase 1 (ULK1), a critical regulator of autophagasome formation, will be examined as a potential drug target with chemical genetics. There are no known inhibitors for this kinase and, therefore, development of a chemical genetic inhibitor system will lend new insight into how ULK1 regulates autophagy and its potential as a drug target.
The proposed research will explore the links between autophagy, a protective cellular process, and cancer. In addition, new chemical tools will be developed for study of the roles and uses for autophagy.
|Garske, Adam L; Peters, Ulf; Cortesi, Arianna T et al. (2011) Chemical genetic strategy for targeting protein kinases based on covalent complementarity. Proc Natl Acad Sci U S A 108:15046-52|