Our recent work has uncovered that tumor-homing peptides that penetrate deep into extravascular tumor tissue contain both a tumor-specific homing sequence and a tissue-penetrating and cell-internalizing C-end Rule (CendR) motif. The CendR element in the tumor-penetrating peptides (TPP) is cryptic and proteolytically activated at the target site. It is not necessary to couple a cargo to the TPP for tumor-selective delivery; free TPP activates a bulk transport pathway in the tumor which carries a co-injected drug or nanoparticle through the vascular wall and deep into the tumor tissue. This application proposes studies to provide detailed understanding of the tissue penetration process that is triggered by TPP. Specifically, we propose: (i) to identify TPP of novel specificities (specific to non-angiogenic tumors and to selected normal organs) using in vivo phage display and de novo design; (ii) to define the molecular pathway of the CendR-induced transport; (iii) to optimize the ability of the peptides to selectively increase tissue permeability; (iv) to validate selected peptide-drug combinations in preclinical treatment and imaging studies. These studies are important for development of CendR-TPP drug targeting platform and lay foundation for the translation of the technology into clinical applications. This proposal is based on our recent discovery of novel cancer targeting compounds: tissue penetrating peptides (TPP; peptide a small protein). These compounds can be used for precision-guided delivery of drugs to cancer blood vessels and deep into tumor tissue. An important aspect of the technology is that TPP do not need to be attached to drugs - it is sufficient to co-inject the drug with a TPP for tumor targeting. Here, we propose to carry out studies to refine and advance the TPP drug del ivery platform. First, we will develop a new generation of TPP that are capable of taking drugs to new targets: dormant tumors and a panel of normal organs. Second, we will define the mechanism of the TPP-mediated targeting pathway and use this understanding to develop optimized regimens for cancer drug delivery. These studies will contribute to establishment of TPP drug targeting platform for cancer targeting and beyond. As TPP can be used to enhance unmodified drugs, a single TPP, once ap proved for clinical applications, can serve as an adjuvant to enhance the activity of various clinically approved drugs and their combinations. As a result, a major advance in targeted drug delivery may ensue.

A complete explanation of many genetic diseases requires that the underlying molecular changes be related to specific changes in cellular events. One example of where this relationship is still not fully understood is how changes at a molecular level ultimately lead to the cellular changes that underlie developmental brain abnormalities such as non-specific X-linked mental retardation (MRX). More than a dozen loci have been implicated in MRX and of the eight genes that have so far been identified, three encode regulators or effectors of the Rho family of small GTPases, so-called 'molecular switches' that regulate signalling pathways in diverse biological processes. These observations are consistent with a growing literature showing that the regulation of Rho GTPase signal transduction pathways are critical for normal neuronal development. For instance, work from a number of laboratories, including the Cline and Van Aelst laboratories at Cold Spring Harbor, has shown that perturbations of the Rho GTPase signal transduction pathways results in abnormal dendrite development. Taken together these lines of evidence support a more general model in which molecular changes that perturb the activity of Rho GTPases could defects in developing neuronal processes that in turn could underlie MRX. The aim of this project is to determine the function of oligophrenin-1, a putative Rho GTPase activating protein (RhoGAP) that is absent in unrelated MRX families. To achieve this aim, I will employ a number of complementary molecular and cellular experimental approaches that are established in the Van Aeslt laboratory to address the following central questions: (i) Where is oligophrenin-1 expressed during the development of mammalian central nervous system? (ii) Which Rho GTPase does oligophrenin-1 act upon in neuronal cells? (iii) What are the effects of the ectopic expression or absence of oligophrenin-1 on cellular morphology of developing neurons? (iv) What proteins does oligophrenin-1 interact with in neurons? More specifically: (i) Polyclonal antibodies will be raised against oligophrenin-1 to determine its expression pattern in the rat developing nervous system. (ii) Biochemical binding assays using the neuronal cell line PC12 will be performed to determine the Rho GTPase target(s) of oligophrenin-1 in neurons. By determining which Rho GTPase oligophrenin-1 acts upon, predictions can be made as to which downstream pathways may be perturbed by loss of this protein in MRX. (iii) Biolistic transfection of organotypic rat hippocampal slices with oligophrenin-1 sense and antisense expression constructs will address the effects of over-expressing and down-regulating oligophrenin-1 levels in hippocampal pyramidal neurons. The detailed dendritic structure of transfected neurons will be imaged using a combination of scanning laser confocal microscopy and two-photon microscopy. These studies will allow the formal testing of the hypothesis that aberrant oliophrenin-1 expression perturbs normal dendrite formation. (iv) The yeast two-hybrid system will be employed to identify oligophrenin-1 interacting proteins in the developing hippocampus. This study will provide further insights into the signalling pathways in which oligophrenin-1 participates as well as identifying additional candidate genes that may be mutated in MRX. Using the combination of molecular and cellular approaches outlined above, I hope to be able to relate specific molecular signalling events to the cellular mechanism underlying MRX.

Completion of the human genome project is expected to reveal that approximately 25% of identified gene sequences will code for membrane proteins containing transbilayer helical domains. This project aims to link the subjects of membrane proteins (including structure, folding and oligomerisation events) with the vast field of lipid properties and dynamics. The work is based on the premise that helical transmembrane proteins fold in two stages, the first stage being helix formation/insertion and the second helix association within the membrane. The second of these stages will be the focus of this study. The interaction of helices within the membrane must involve changes in lipid-lipid interaction and lipid-helix interactions as well as helix-helix interactions. The proposed work will focus on the influences of the lipid environment on folding and oligomerisation events of a-helical membrane proteins. Helix-helix association constants in lipid bilayers will be measured with the aim of observing changes in the association constant resulting from changes in the lipid environment. The transmembrane domain of human glycophorin A dimerises in both detergent and lipid bilayer environments. It is this process that will be studied in lipid vesicle systems. Various lipid properties, such as bilayer thickness, lipid lateral pressure and bilayer stiffness can be controlled by changes in the composition of the host membrane system. The host membranes will be composed of biologically relevant phospholipids along with cholesterol, which is also prevalent in biological membranes. The physical properties of the host membrane will be determined by separate measurements. Helix association will be measured using Forster Resonance Energy Transfer with a donor and acceptor complex on the N-terminus of the synthetic glycophorin A peptide. Helix association will be measured as a function of specific physical membrane properties. A second technique that has recently been developed in the Engelman lab will also be used. This is a new genetic assay for helix association, which allows studies in the inner membrane of living E.coli. The function of many transmembrane proteins have been shown to be modulated by lipid composition. These studies will provide important information in this area along with other fields of study such as transmembrane signalling events (which often involve changes in protein association) and selective transport in membrane trafficking (where some models postulate bilayer thickness effects to segregate retained and transported proteins).

Our recent work has uncovered that tumor-homing peptides that penetrate deep into extravascular tumor tissue contain both a tumor-specific homing sequence and a tissue-penetrating and cell-internalizing C-end Rule (CendR) motif. The CendR element in the tumor-penetrating peptides (TPP) is cryptic and proteolytically activated at the target site. It is not necessary to couple a cargo to the TPP for tumor-selective delivery; free TPP activates a bulk transport pathway in the tumor which carries a co-injected drug or nanoparticle through the vascular wall and deep into the tumor tissue. This application proposes studies to provide detailed understanding of the tissue penetration process that is triggered by TPP. Specifically, we propose: (i) to identify TPP of novel specificities (specific to non-angiogenic tumors and to selected normal organs) using in vivo phage display and de novo design; (ii) to define the molecular pathway of the CendR-induced transport; (iii) to optimize the ability of the peptides to selectively increase tissue permeability; (iv) to validate selected peptide-drug combinations in preclinical treatment and imaging studies. These studies are important for development of CendR-TPP drug targeting platform and lay foundation for the translation of the technology into clinical applications. This proposal is based on our recent discovery of novel cancer targeting compounds: tissue penetrating peptides (TPP; peptide a small protein). These compounds can be used for precision-guided delivery of drugs to cancer blood vessels and deep into tumor tissue. An important aspect of the technology is that TPP do not need to be attached to drugs - it is sufficient to co-inject the drug with a TPP for tumor targeting. Here, we propose to carry out studies to refine and advance the TPP drug del ivery platform. First, we will develop a new generation of TPP that are capable of taking drugs to new targets: dormant tumors and a panel of normal organs. Second, we will define the mechanism of the TPP-mediated targeting pathway and use this understanding to develop optimized regimens for cancer drug delivery. These studies will contribute to establishment of TPP drug targeting platform for cancer targeting and beyond. As TPP can be used to enhance unmodified drugs, a single TPP, once ap proved for clinical applications, can serve as an adjuvant to enhance the activity of various clinically approved drugs and their combinations. As a result, a major advance in targeted drug delivery may ensue.