Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity. We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage. We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ. Different peptide motifs were recovered from each of these tissues. The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold. Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail. Immunohistochemistry showed that the phage localized in the blood vessels of their target organ. When tested, the phage homing was blocked in the presence of the cognate peptide. By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ-and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon. Our results also show that these molecular differences can serve as molecular addresses. ( J. Clin. Invest.

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10 -15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancerprone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk. Diseases affecting the prostate have gained major significance clinically and economically, primarily because of the increasing average age of the male population in the industrialized countries. Benign prostate hyperplasia affects to some degree most elderly men. Even more serious, the prostate is a frequent site of cancer. Some autopsy studies find that most men older than 70 have occult or overt cancer in the prostate (1). The surgical therapies of prostate hypertrophy and prostate cancer are associated with serious side effects, such as incontinence and impotence.We have sought to develop a strategy that would provide a less traumatic treatment for prostate disease than is currently available. Our strategy is based on identification of peptides that home to specific sites in the vasculature by in vivo screening of intravenously injected phage libraries. These studies have revealed a surprising degree of specialization in the endothelia of various normal tissues (2, 3). Screening phage libraries for tumor homing has yielded a collection of peptides that home to tumor vasculature (4). We and others have used these tumor-homing peptides to direct therapies into tumors in mice (4, 5). We report here the identification of peptides that home to the vasculature of the prostate and the use of one of these homing peptides to deliver a proapoptotic peptide to the prostate. Materials and MethodsMaterials. Peptides were synthesized to our specifications by AnaSpec (San Jose, CA) or by our Peptide Synthesis Facility. The peptides were purified by HPLC and their identity was confirmed with mass spectrometry.Apotag Kit for TUNEL staining was purchased from Intergen (Purchase, NY). Testosterone pellets (12.5 mg) and control pellets were from Innovative Research of America (Sarasota, FL), and controlled release pumps ...

The vasculature of individual tissues is highly specialized. The endothelium in lymphoid tissues expresses tissue-specific receptors for lymphocyte homing, and recent work utilizing phage homing has revealed an unprecedented degree of specialization in the vasculature of other normal tissues. In vivo screening of libraries of phage that displace random peptide sequences on their surfaces has yielded specific homing peptides for a large number of normal tissues. The tissue-specific endothelial molecules to which the phage peptides home may serve as receptors for metastasizing malignant cells. Probing of tumor vasculature has yielded peptides that home to endothelial receptors expressed selectively in angiogenic neovasculature. These receptors, and those specific for the vasculature of individual normal tissues, are likely to be useful in targeting therapies to specific sites.

In vivo phage display is a powerful method to study organ-and tissue-specific vascular addresses. Using this approach, peptides capable of tissue-specific homing can be identified by performing a selection for that trait in vivo. We recently showed that the CGFECVRQCPERC (termed GFE-1) peptide can selectively bind to mouse lung vasculature after an intravenous injection. Our aim in the present study was to identify the receptor for this lung-homing peptide. By using affinity chromatography, we isolated a 55-kDa lung cell-surface protein that selectively binds to the GFE-1 peptide. Protein sequencing established the identity of the receptor as membrane dipeptidase (MDP), a cell-surface zinc metalloprotease involved in the metabolism of glutathione, leukotriene D 4 , and certain ␤-lactam antibiotics. Phage particles displaying the GFE-1 peptide selectively bind to COS-1 cells transfected with the murine MDP cDNA. Moreover, the synthetic GFE-1 peptide could inhibit MDP activity. By establishing MDP as the receptor for the GFE-1 peptide, our results suggest potential applications for both MDP and the GFE-1 peptide in delivery of compounds to the lungs. This work also demonstrates that cell-surface proteases can be involved in tissuespecific homing.Phage display libraries are used to obtain defined peptide sequences interacting with a particular molecule (1-5). We recently used phage display libraries to select in vivo for peptides capable of homing to the vasculature of a given organ or tissue (6-8). This approach consists of intravenously injecting a phage library into a mouse, allowing the phage to circulate in vivo, and then collecting phage bound to the vascular bed of interest. By using this strategy, we identified phage-displayed peptides capable of homing to the vasculature of brain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland and prostate 1 (6, 7). We also identified peptides that selectively home to blood vessels of experimental tumors in nude mice (8). When coupled to the anti-cancer drug doxorubicin, these tumor-homing peptides enhanced the efficacy of doxorubicin against human breast cancer xenografts and reduced its overall toxicity (8).There are many reports of tissue-specific interactions between blood cells and endothelial cells. Most of the cases studied at a molecular level involve lymphocyte homing and are generally mediated by classical cell-cell adhesion molecules (9). Another example of vascular bed-specific recognition is the preference of metastasizing tumor cells for certain organs (10, 11). In the lung, several vascular receptors mediate adhesion of metastatic cells. For example, Elble et al. (12) showed that lung endothelial cell adhesion molecule-1, an endothelial surface protein with sequence homology to chloride channels, mediates adhesion of malignant melanoma cells to lung endothelium. The same group also showed that a protease, lung endothelial dipeptidyl peptidase IV, is responsible for homing of metastatic breast and prostate carcinoma cells to lung (13). These ...

The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed of two chains, α and βc. Both chains belong to the superfamily of cytokine receptors characterized by a common structural feature, i.e., the presence of at least two fibronectin-like folds in the extracellular domain, which was first identified in the growth hormone receptor. The GM-CSF receptor (GMR)-α chain confers low affinity binding only (5–10 nM), whereas the other chain, βc, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-α (25–100 pM). The present study was designed to define the assembly of the GMR complex at the molecular level through site-directed mutagenesis guided by homology modeling with the growth hormone receptor complex. In our three-dimensional model, R280 of GMR-α, located in the F′–G′ loop and close to the WSSWS motif, is in the vicinity of the ligand Asp112, suggesting the possibility of electrostatic interaction between these two residues. Through site directed mutagenesis, we provide several lines of evidence indicating the importance of electrostatic interaction in ligand–receptor recognition. First, mutagenesis of GMR-αR280 strikingly ablated ligand binding in the absence of β common (βc); ligand binding was restored in the presence of βc with, nonetheless, a significant shift from high (26 pM) toward low affinity (from 2 to 13 nM). The rank order of the dissociation constant for the different GMR-αR280 mutations where Lys > Gln > Met > Asp, suggesting the importance of the charge at this position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10–20-fold). Third, removal of the charge at position R280 of GMR-α introduced a 10-fold decrease in the association rate constant and only a 2-fold change in the dissociation rate constant, suggesting that R280 is implicated in ligand recognition, possibly through interaction with Asp112 of GM-CSF. For all R280 mutants, the half-efficient concentrations of GM-CSF required for membrane (receptor binding) to nuclear events (c-fos promoter activation) and cell proliferation (thymidine incorporation) were in the same range, indicating that the threshold for biologic activity is governed mainly by the affinity of ligand–receptor interaction. Furthermore, mutation of other residues in the immediate vicinity of R280 was less drastic. Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a β turn in a highly divergent context at the F′–G′ loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

The receptor for the hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of two chains, both of which belong to the superfamily of cytokine receptors. The ␣ chain confers low affinity binding only, whereas the ␤ chain (␤ c ) confers high affinity binding when associated with ␣. Ectopic expression of both chains of the receptor in murine NIH-3T3 fibroblasts results in signal transduction, mitogenesis, and morphologic transformation. The cytoplasmic domain of the GM-CSF receptor ␣ subunit (GMR-␣) comprises 54 amino acids that have been shown to be important for signal transduction through the ␤ chain. The present study was designed to address the possibility of receptor oligomerization and its functional implication. Cross-linking studies with 125 I-GM-CSF on NIH-3T3 transfectants is consistent with the presence of ␣ and ␤ c dimers and of receptor oligomers. We have, therefore, generated an inert ␣ chain through polymerase chain reaction-mediated truncation of 47 amino acids of the COOH-terminal domain of ␣ (␣ t ), and coexpressed ␣ t , ␣, and ␤ c in NIH-3T3. In cells in which ␣ t and ␣ are present in stoichiometric proportion within the GM-CSF-binding complex, we provide evidence that ␣ t is dominant negative over wild type ␣ on the basis of two different functional assays: cell proliferation and foci formation. Hence, our results suggest the requirement for at least two functional ␣ chains for signal transduction. Together with the cross-linking studies, our data indicate that the functional GMR is an oligomer that contains at least two ␣ chains.

tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V. In vitro selection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

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