Alpha Helix Mimetics

Abstract

Proteins mediate biological functions through exquisite specificities for their macromolecular targets, but their uses in biology and medicine are compromised by their high cost of manufacture and low bioavailability. The objective of this thesis is to downsize proteins to small, constrained, a-helical peptides with stable structures in water and potent biological activities. The thesis presents studies of biologically important peptide sequences chemically constrained into a-helical structures using one or more (iri+4) side chain to side chain bridge constraints. Proteins typically use 2-4 turns (l 15 residues) of an a-helix to interact with other proteins and nucleotides. However short peptides of this size are not thermodynamically stable helices in water and usually unwind to random structures that have little or no biological activity. By contrast, strategically helix-constrained short peptides are shown herein to have very stable a-helical structures in water and this structure induction translates into 10-1000 fold higher affinity and/or biological activity than for unconstrained peptides of the same sequence. Enforcing the a-helical structure in short peptides derived from viral (HIV), bacterial (CSP) and human (Nociceptin, Bad) proteins has produced similar biological properties to those observed for the native proteins bearing the same helical components. These observations suggest that the general approach of mimicking protein helices in much smaller molecules could have many promising applications in chemistry, biology and medicine. In Chapter 2, an RNA-binding 17-residue helical peptide fragment of the transporter protein HIV-1 Rev is investigated, this being important for HIV replication. Since this fragment has no structure in water and yet is a-helical when bound to RNA, the helix-inducing ability of a number of N-terminal caps connected to this peptide was investigated. Positioning an (i ri+4) lactam bridge constraint at the N-terminus was more effective than within the sequence for enhancing helical structure and RNA-affinity due to steric interference. In particular, Ac-(1,5- cyclo)-[KARAD]AAA- attached to the N-terminus of this peptide induced the most a-helicity and improved binding affinity for a segment of RNA (the Rev Responsive Element), overcoming an entropic cost estimated at ~17.7 kJ/mol.K. Chapter 3 examines agonist activity for a 17-residue peptide fragment of nociceptin that activates the ORL-1 G protein coupled receptor and has role in sensory perception and pain. Nociceptin has an unstructured ltriggering or messager N-terminus thought to bind and activate in the transmembrane region of ORL-1, as well as a C-terminal a-helical laddressr domain thought to bind the extracellular surface of ORL-1. Different K(i) r D(i+4) lactam bridge constraints within nociceptin(1-17)-NH2 had a significant influence on activation of the receptor, due either to the effect of amino acid replacement or to steric interactions with ORL-1. Importantly, appropriately positioned helix constraints promoted a-helicity in the C-terminus while retaining the unstructured nature of the N-terminus, leading to up to 20 and 4 fold increased agonist and antagonist potency, respectively. Chapter 4 reports a study of a 17-residue Competence Stimulating Peptide (CSP-1) from Streptococcus pneumoniae CSP-1, a quorum sensing peptide that has been shown to have antibacterial potential when used at concentrations higher than those required for competency and bacterial growth. Use of the helix constraints induced a-helicity in the CSP-1 sequence in aqueous environments, as shown by CD spectra and an NMR-derived solution structure, whereas the native sequence had no structure in water. The constrained peptide had g10 fold higher anti-bacterial activity than unconstrained CSP-1 peptide against S. pneumoniae, consistent with helix induction being important for binding to its receptor, claimed to be a histidine kinase. Chapter 5 describes the effects of helix constraints in the BH3 peptide Bad on binding to antiapoptotic proteins Bcl-xL. Placing helix constraints at different positions within the peptide resulted in quite different degrees of helicity and affinity for Bcl-xL. This library of compounds demonstrated that a particular segment within Bad, the BH3 domain that is conserved in many other apoptotic proteins, needs to be a-helical for binding and activity. Cytotoxicity and cell death were assessed in vitro using B cell lymphocytes. We showed that an a-helical BH3 peptide could bind with moderate affinity to Bcl-xL and, like its parent Bad, was able to induce cytochrome c release from isolated mitochondria, consistent with the ability to induce apoptosis. Chapter 6 summarizes the key conclusions and implications, including correlations between structure and biological activity, as well as suggesting potential directions for future work aimed at exploiting helical peptidomimetics.