Therapeutic cancer targeting peptides

Antitumor monoclonal antibodies have shown clinical promise as cancer cell surface targeting agents. More tumor targeting antibodies are likely to be approved by the FDA in the next few years. However, there are two major limitations in antibody-targeted therapy: large size and nonspecific uptake of the antibody molecules by the liver and the reticuloendothelial system. These result in poor tumor penetration of antibody pharmaceuticals and dose-limiting toxicity to the liver and bone marrow. Peptides are excellent alternative targeting agents for human cancers, and they may alleviate some of the problems with antibody targeting. In the last decade, several investigators have successfully used combinatorial library methods to discover cell surface binding peptides that may be useful for cancer targeting. The phage-display library technique and the "one-bead one-compound" combinatorial library method are the two approaches that have been used. Cancer cell surface receptors or endothelial cell surface receptors of the neovasculature are the two popular therapeutic targets for cancer. Results from preclinical studies with some peptides are encouraging in their targeting potential.     

Identification of a novel human Acyl-CoA binding protein isoform with a unique C-terminal domain

Seven isoforms of the multifunctional human Acyl-coenzyme A binding protein (ACBP) have been characterized so far. Through ab initio analysis of expressed sequence tag (ESTs), we identified a novel high-abundant ACBP splice variant ACBP1e encoding an ACBP isoform with a unique C-terminus of 81 amino acid residues. Bioinformatic analysis shows that this domain is evolutionary conserved and shares no significant homology with other known proteins, and its function is not known. Quantitative RT-polymerase chain reaction (PCR) revealed that ACBP1e is predominantly expressed in adipose tissue and hippocampus. Protein expression studies showed perinuclear clustering of ACBP1e. These clusters were not seen in ACBP1e mutants with an altered putative subtilisin/kexin isozyme-1 cleavage site within the C-terminus, indicating that this domain is required for proper localization of ACBP1e. Conclusively, we identified a novel ACBP isoforms with an unique C-terminal domain encoded by a high-abundant splice variant.     

Therapeutic targeting of cancer cell cycle using proteasome inhibitors

Proteasomes are multicatalytic protease complexes in the cell, involved in the non-lysosomal recycling of intra-cellular proteins. Proteasomes play a critical role in regulation of cell division in both normal as well as cancer cells. In cancer cells this homeostatic function is deregulated leading to the hyperactivation of the proteasomes. Proteasome inhibitors (PIs) are a class of compounds, which either reversibly or irreversibly block the activity of proteasomes and induce cancer cell death. Interference of PIs with the ubiquitin proteasome pathway (UPP) involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI induced cell cycle arrest in a variety of cancer cells. Although many PIs target the proteasomes, not all of them are effective in cancer therapy. Some cancers develop resistance against proteasome inhibition by possibly activating compensatory signaling pathways. However, the details of the activation of these pathways and their contribution to resistance to PI therapy remain obscure. Delineation of these pathways may help in checking resistance against PIs and deducing effective combinational approaches for improved treatment strategies. This review will discuss some of the signaling pathways related to proteasome inhibition and cell division that may help explain the basis of resistance of some cancers to proteasome inhibitors and underline the need for usage of PIs in combination with traditional chemotherapy.     

The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase

The rod outer segment ATP binding cassette (ABC) transporter protein (ABCR) plays an important role in retinal rod cells presumably transporting retinal. Genetic studies in humans have linked mutations in the ABCR gene to a number of inherited retinal diseases particularly Stargardt macular degeneration and age-related macular degeneration (ARMD). The ABCR protein is characterized by two nucleotide binding domains and two transmembrane domains, each consisting of six membrane-spanning helices. We have cloned and expressed the 376 amino acid (aa) C-terminal end of this protein (amino acid residues 1898-2273) containing the second nucleotide binding domain (NBD2) with a purification tag at its amino terminus. The expressed protein was found to be soluble and was purified using a rapid and high-yield single-step procedure. The purified protein was monomeric and migrated as a 43 kDa protein in SDS-PAGE. The purified NBD2 protein had strong ATPase activity with a K(m) of 631 microM and V(max) of 144 nmol min(-1) mg(-1). This ATPase activity on normalization was kinetically comparable to that observed for purified and reconstituted native ABCR. Nucleotide inhibition studies suggest that the binding of NBD2 is specific for ATP/dATP, and that none of the other ribonucleotides appeared to compete for binding at this site. These studies demonstrate that cloned and expressed NBD2 protein is a fully functional ATPase in the absence of the remainder of the molecule. The level of ATPase activity was comparable to that of trans-retinal-stimulated ABCR ATPase. The NBD2 expression plasmid was used to generate a Leu2027Phe mutation associated with Stargardt disease. Analysis of the ATPase activity of the mutant protein demonstrated that it had a 14-fold increase in binding affinity (K(m) = 46 microM) with a corresponding 9-fold decrease in the rate of hydrolysis (V(max) = 16.6 nmol min(-1) mg(-1)), indicating a significant alteration of the ATPase function. It also provided a molecular basis of Stargardt disease involving this mutation.     

Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302)

In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.     

Therapeutic targeting of cytokines

Over recent years, with many advances in understanding the pathological processes that underlie many inflammatory conditions, it has become clear that targeting cytokines has proven to be highly effective therapeutically. The second International Cytokine symposium, held recently in Manchester UK, has provided an important forum for bringing basic and clinical scientists together to discuss many aspects of cytokine medicine from the laboratory to the clinic. In this article, we provide an overview of the main issues raised from this important conference and set the scene for more detailed reports that follow in subsequent articles in this journal.     

Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan

Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.     

DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 2: C-terminal benzimidazoles and derivatives

The synthesis and in vitro potency of DNA minor-groove binding antibacterials lacking the C-terminal amide bond are described. The crescent shaped molecules bear the positively charged amino group at an internal pyrrole unit instead of the C-terminus. Three structural parameters were investigated: the N-terminal unit, the internal amino group, and the C-terminal ring system. Several compounds demonstrated good in vitro potency against various Gram-positive bacteria and some molecules were moderately active against Escherichia coli, a representative Gram-negative strain.     

Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.     

C-terminal arginine cluster is essential for receptor binding of norovirus capsid protein

Noroviruses are the major viral pathogens of epidemic acute gastroenteritis affecting people worldwide. They have been found to recognize human histo-blood group antigens as receptors. The P domain of norovirus capsid protein was found to be responsible for binding to viral receptors, and the recombinant P protein forms P dimers and P particles in vitro. In this study, we demonstrate that a highly conserved arginine (R) cluster at the C terminus of the P domain is critical for receptor binding and P particle formation of the P proteins. Deletions of the R cluster abolished these functions. Replacement of the R cluster with histidines (another positively charged amino acid) resulted in low efficiency of receptor binding and P particle formation, while replacement with alanines led to loss of both functions completely. The R cluster also contains a highly conserved trypsin digestion site. A treatment of capsid protein or P domain mutants from both genogroup I (Norwalk virus) and genogroup II (VA387) noroviruses with trypsin resulted in a removal of the R cluster and the S domain, leaving a P polypeptide of 31.3 kDa (Norwalk virus) or 34.3 kDa (VA387), similar to the soluble P protein found in vivo. Our findings imply that the proteolytic process could be a necessary step for norovirus replication in the host.     

Specificity and promiscuity in human glutaminase interacting protein recognition: insight from the binding of the internal and C-terminal motif

A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO(-)) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH(2)COO(-)) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics.     

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.     

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.