Selection of peptide aptamers that recognize and inhibit intracellular proteins

Most cellular processes are governed by complex genetic regulatory networks. These networks consist of large numbers of genes whose protein products show intricate patterns of genetic and physical interactions with each other. Understanding how these networks operate inside cells to affect cellular decisions will require tools to analyze the interactions among network members. This thesis describes the development of a new class of molecules designed to disrupt the interactions between members of genetic regulatory networks, and inhibit protein function.; A library of conformationally constrained peptides, displayed in the active site of Escherichia coli Thioredoxin (TrxA), was expressed in yeast. A two-hybrid approach was then used to select those that interacted with human Cyclin-dependent kinase 2 (Cdk2), a serine/threonine kinase required for progression through the G1 phase of the cell cycle. These peptide aptamers bound Cdk2 tightly and specifically with equilibrium dissociation constants in the nanomolar range. When expressed and purified from bacteria, some peptide aptamers inhibited the kinase activity of Cdk2 in an in vitro assay. These results suggested that, similar to monoclonal antibodies, peptide aptamers can recognize protein targets with high affinity and high specificity, and by doing so can inhibit the function of their protein targets.; A further characterization of one particular Cdk2 interacting aptamer, pep8, was undertaken. Unlike naturally occurring Cdk inhibitors such as p21{dollar}sp{lcub}rm Cip1{rcub}{dollar} which inhibit the activity of Cdk2 on all its substrates, the inhibition by pep8 shows distinct substrate specificity. Further experiments demonstrated that pep8 is a competitive inhibitor of Cdk2 that binds at or near the active site. Expression of pep8 in human cells retards their progression thorough the G1 phase of the cell cycle. Taken together these results suggested that pep8 inhibits cell cycle progression by blocking the activity of Cdk2 on a subset of its substrates.; The peptide aptamer approach was extended to the Human Immunodeficiency Virus Type 1 (HIV-1) Rev protein. Some Rev interacting aptamers discriminated between different wildtype alleles of Rev, demonstrating that peptide aptamers are capable of very specific molecular recognition. The aptamers all bound to the N-terminal domain of Rev suggesting that peptide aptamers show distinct biases for certain structural features on protein surfaces. The results with Rev show that peptide aptamers that recognize a variety of different proteins can be isolated.; An affinity maturation protocol was developed to allow tighter binding variants of peptide aptamers to be selected from mutagenized pools. This method was used both to evolve a single Cdk2 interactor into a tighter binding variant, and to select tighter binders from a pool of aptamers that interacted with Rev. The results suggest that affinity maturation will be useful for generating high affinity peptide aptamers against a variety of targets, and should expand the usefulness of peptide aptamers as inhibitors of protein function.