Current Drug Discovery Technologies - Volume 5, Issue 1, 2008

Selective activation of blood coagulation in tumor vessels with subsequent thrombosis and tumor infarction is a promising strategy in cancer therapy. To this end, different fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) were fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or cyclic derivates of these peptides, which selectively target αv-integrins or aminopeptidase N (CD13), respectively. Rationale for this strategy is the fact that these surface receptors are preferentially expressed on tumor endothelial cells. The tTF constructs were expressed in Escherichia coli BL21 (DE3). The integrity of the fusion proteins was evaluated by SDS-PAGE, immunoblotting and mass spectrometry. The screening process for the activity contained coagulation assays as well as purified receptor binding assays. The fusion proteins which retained their thrombogenic and binding activity were evaluated further. In vivo studies in nude mice bearing established different malignant human tumors revealed that i.v. administration of tTF-RGD or tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels, which was demonstrated by histological analysis. Furthermore, treatment studies showed that the targeted tTF fusion proteins but not untargeted tTF proteins induced significant tumor growth retardation in human adenocarcinoma of the breast in a nude mice model without apparent side effects such as thrombosis in liver, kidney, heart or lung at therapeutic dose levels. Finally, we illustrate the upscaling process of fusion protein fabrication in order to produce the amounts needed for clinical studies. Thus, generation and screening of active fusion proteins, which induce selective thrombosis in the tumor vasculature, may be a promising strategy for the development of new drugs as cancer therapeutics.

In recent years, significant progresses have been achieved in clinical oncology due to a plethora of new chemotherapeutic drugs and agents for targeted therapy. However, clinical response and overall survival rates have not improved significantly for a large number of different tumors. This may at least in part be due to the enormous genetic heterogeneity among tumors, even within a tumor entity. Moreover, besides individual somatic mutations or combinations of these in malignant tumors, the genetic background of each individual cancer patient appears to have a major impact on treatment response and overall survival. Current DNA microarray technology allows the simultaneous gene expression analysis of all known genes, and large-scale gene expression studies have provided new molecular classifications for a series of different tumors. Evidence has also been provided that gene expression signatures of malignant tumors may predict treatment response to classical chemotherapeutic or targeted anticancer drugs. The presented report summarizes the current knowledge about the role of gene expression signatures as putative guides for treatment decisions, with the future prospect of individualized treatment approaches for cancer patients.

Despite recent advances in the diagnoses and treatment of breast cancer, this disease continues to be a major cause of death. One of the biggest challenges in breast cancer treatment is bone metastasis. Breast cancer cells (BCCs) are capable of migrating to the bone marrow and utilizing the marrow microenvironment to remain quiescent. While exhibiting quiescence in the marrow, BCCs can evade the effects of conventional cancer treatments such as chemotherapy. Therefore, scientists must find a new paradigm to target these quiescent BCCs. The development of potential targets may require a more comprehensive understanding of the marrow microenvironment and its regulators. The preprotachykinin-1 (PPT-I) gene encodes for the tachykinin peptides, which interact with neurokinin (NK) receptors. Studies have correlated this interaction with BCC integration into the bone marrow and breast cancer progression. In this review, we discuss the roles that different factors of the marrow microenvironment play in breast cancer and targets of NK receptors as potential treatment options.

Components of the cell-surface and extracellular matrix (including cell walls of some microorganisms) present ideal but often challenging targets for disease detection and targeting. In the past few years, selection of peptide and oligonucleotide biomimics against secreted and extracellular targets has resulted in the isolation of small high affinity binding agents against target molecules. However, little attention has been paid to the interaction of these biomimics with glycans (specifically, cell surface glycans) despite their ubiquitous and abundant presence on every cell (glycocalyx). Although glycans are now accepted as critically important class of molecules as biomarkers, imaging, therapeutic and drug targets, little advance has been made in exploiting these molecules for clinical applications. The diversity and complexity of glycan structures along with time-consuming analytical and synthetic methods are among the most significant challenges faced by glycoscientists. Innovative technologies are urgently needed to overcome these challenges and to develop glycan- based clinical applications. This review article will provide an overview of existing biocombinatorial methods that focus on the selection and use of glycan-binding random oligonucleotide ligands (aptamers) as conceptually novel therapeutics for a variety of biomedical applications including anti-microbial and anti-cancer agents. Given the range and importance of protein-carbohydrate interactions in biological processes, rapid discovery of agents that mediate these interactions may provide fruitful venues towards novel therapeutics.

The Toll-like receptor (TLR) family plays a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection. TLRs recognize distinct microbial components and activate intracellular signaling pathways that induce expression of host inflammatory genes. Extensive research in the past decade to understand TLR-mediated mechanisms of innate immunity has enabled pharmaceutical companies to begin to develop novel therapeutics for the purpose of controlling inflammatory disease. Initially, extracellular TLR agonists were designed to compete with natural microbial ligands for binding to TLRs. More recently, basic research to identify new targets for drug development has begun to explore modulation of TLR intracellular signaling pathways, in addition to TLR ligand binding. In this review, we will discuss recent strategies, including the use of decoy peptides and mimetics, plant polyphenols, and chemically modified antisense oligonucleotides, that inhibit different molecular events in TLR signaling pathways to modulate the inflammatory response. The molecular mechanisms of these inhibitors range from interference with protein-protein interactions between signaling proteins, to inhibition of transcription factor activity, to perturbation of the plasma membrane, and are derived from host, pathogen, and plant sources and by rational design. Taken together, these studies represent promising avenues for the development of novel tailored immune therapeutics that can relieve the great toll inflicted by inflammatory disease on human health and quality of life.

Site-specific drug delivery to bone is considered to be achievable by utilizing acidic amino acid homopeptides. We found that fluorescence-labeled acidic amino acid (L-Asp or L-Glu) homopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged an enzyme, tissue-nonspecific alkaline phosphatase, to see whether this would improve the efficacy of enzyme replacement therapy. The L-Asp hexapeptide-tagged drugs, including the enzyme, were selectively delivered to bone in comparison with the untagged drugs. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug or enzyme from the acidic oligopeptide. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged estradiol and levofloxacin, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly removed by hydrolysis in the body after delivery to bone. The adverse effect of estradiol on the uterus was greatly reduced by conjugation to the hexapeptide. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs and enzyme.

MicroRNAs (miRNAs) are endogenous, short, double-stranded and noncoding RNA molecules that have been identified in a variety of organisms and certain viruses. This group of new molecules is transcribed mainly from the introns and/or exons or intergenic regions and plays important regulatory roles in development and gene expression. Mature miRNAs are typically 20-24 nucleotides in length and regulate target mRNAs post transcriptionally by interactions with partially mismatched sequences in the 3’untraslated regions of these messengers. These interactions result in the suppression of translation or degradation of target mRNAs. At the present, although the biological functions of miRNAs are not completely revealed, a growing body of evidence implicates that miRNA pathway is a new mechanism of gene regulation in both normal and diseased conditions and therefore investigation of miRNA biogenesis and function may add new tools for gene functional study and drug development. In this article, we will briefly review the structure, biogenesis and basic mechanism of action of miRNAs identified in higher organisms and viruses and then focus on the recent progress in research for drug development using the miRNA pathway as a strategy. Particularly, we will discuss the advance, challenge and future directions on antiviral drug development using miRNA as a target or a gene silencing tool for the treatment of viral infections.

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.

Ligand-bound and free structures of brain membrane targets for L-glutamate (Glu) suggest the view, that quaternary rearrangements are associated with ligand binding. Rearrangement of the machinery of the signaling apparatus, such as molecular switches, recognition sites and the target structures for ligand binding of Glu-operated ion channel and heptahelical G-protein-coupled family receptors have been quantified and compared with the use of the root mean square (RMS) values. In addition to conformational rearrangement of the Glu receptor structures in complex with a series of ligands, conformations of Glu in various target structures became available. High resolution data revealed that the extended Glu conformation is conserved in the binding crevice of all ionotropic Glu receptors (iGluRs). Furthermore, the extended conformations of Glu that characterize iGluRs and mGluRs are distinguishable by distance and torsion angle parameters, such as δC1-C2 and Cα-Cβ-Cγ-C2. By contrast, a bent Glu conformation is recognized in Glu transporters.

Accumulating evidence implicates a failure of myelin-reactive immune cells to undergo apoptosis in the pathological events contributing to multiple sclerosis (MS). We have recently demonstrated that members of the inhibitor of apoptosis (IAP) family of antiapoptotic genes are elevated in peripheral blood immune cells (monocytes, T cells) of patients with aggressive forms of MS (secondary progressive) or those with relapsing-remitting MS suffering a disease replase. These findings suggest that the IAPs may be novel diagnostic markers for distinguishing subtypes of MS. Moreover, antisense-mediated knockdown of the IAP family member known as Xlinked IAP (XIAP) reverses paralysis in an animal model of MS suggesting that treatments targeting XIAP, and perhaps other IAPs, may have utility in the treatment of MS.

Growth factor-activated receptor tyrosine kinases (RTKs) undergo rapid endocytosis and degradation in lysosomes. This process, known as receptor downregulation, is essential to prevent the overgrowth of cells by terminating signal transduction from activated RTKs. Thus, defects in RTK downergulation lead to cell proliferative disorders such as cancer. Upon endocytosis, RTKs are delivered to endosomes, from where they are further transported to lysosomes. Ubiquitin serves as a sorting signal that is tagged on activated RTKs and directs their trafficking from endosomes to lysosomes. On the endosomal membrane, ubiquitinated RTKs are sorted by coordinated actions of the class E vacuolar protein sorting (Vps) proteins some of which form complexes that directly recognize the ubiquitin moieties of RTKs. UBPY and AMSH in mammals, as well as Doa4 in yeast, are deubiquitinating enzymes (DUBs) that associate with class E Vps proteins on endosomes. Here I review the recently unveiled roles and regulatory mechanisms of these DUBs in the endosomal sorting of ubiquitinated cargo proteins. These findings suggest that RTK downregulation is controlled not only by ubiquitination but also by deubiquitination of RTKs as well as other endosomal proteins. Therefore, elucidating the entire functions and regulation of the endosomal DUBs potentially provides novel molecular targets for the treatment of cancer accompanied by overexpression or constitutive activation of RTKs.