Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques

This review begins with a general presentation of the new paradigm of drug discovery, with its emphasis on the rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties. The focus of the paper is on the various experimental methods used to determine such key physicochemical properties as ionization, lipophilicity and distribution in isotropic and anisotropic systems, solubility, and permeability across artificial membranes. Both traditional and high-throughput methods are presented and their limits highlighted. The text concludes with the trade-off between quantity/speed in high-throughput screening techniques versus greater data quality in the more labor-intensive methods. Received 23 April 2002; received after revision 25 June 2002; accepted 11 July 2002 RID="*" ID="*"Corresponding author.     

Cell-penetrating peptides: tools for intracellular delivery of therapeutics

The main problem of therapeutic efficiency lies in the crossing of cellular membranes. Therefore, significant effort is being made to develop agents which can cross these barriers and deliver therapeutic agents into cellular compartments. In recent years, a large amount of data on the use of peptides as delivery agents has accumulated. Several groups have published the first positive results using peptides for the delivery of therapeutic agents in relevant animal models. These peptides, called cell-penetrating peptides (CPPs), are short peptides (fewer than 30 residues) with a net positive charge and acting in a receptor- and energy-independent manner. Here, we give an extensive review of peptide-mediated delivery systems and discuss their applications, with particular focus on the mechanisms leading to cellular internalization.Received 14 March 2005; received after revision 25 April 2005; accepted 28 April 2005     

Targeted polymeric micelles for delivery of poorly soluble drugs

Polymeric micelles (micelles formed by amphiphilic block copolymers) demonstrate a series of attractive properties as drug carriers, such as high stability both in vitro and in vivo and good biocompatibility, and can be successfully used for the solubilization of various poorly soluble pharmaceuticals. These micelles can also be used as targeted drug delivery systems. The targeting can be achieved via the enhanced permeability and retention effect (into the areas with the compromised vasculature), by making micelles of stimuli-responsive amphiphilic block copolymers, or by attaching specific targeting ligand molecules to the micelle surface. Immunomicelles prepared by coupling monoclonal antibody molecules to p-nitrophenylcarbonyl groups on the water-exposed termini of the micelle corona-forming blocks demonstrate high binding specificity and targetability. Immunomicelles prepared with cancer-specific monoclonal antibody 2C5 specifically bind to different cancer cells in vitro and demonstrate increased therapeutic activity in vivo. This new family of pharmaceutical carriers can be used for the solubilization and targeted delivery of poorly soluble drugs to various pathological sites in the body.     

Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited

Clustering of neurotransmitter receptors in the postsynaptic membrane is critical for efficient synaptic transmission. During neuromuscular synaptogenesis, clustering of acetylcholine receptors (AChRs) is an early sign of postsynaptic differentiation. Recent studies have revealed that the earliest AChR clusters can form in the muscle independent of motorneurons. Neurally released agrin, acting through the muscle-specific kinase MuSK and rapsyn, then causes further clustering and localization of clusters underneath the nerve terminal. AChRs themselves are required for agrin-induced clustering of several postsynaptic proteins, most notably rapsyn. Once formed, AChR clusters are stabilized by several tyrosine kinases and by components of the dystrophin/utrophin glycoprotein complex, some of which also direct postnatal synaptic maturation such as formation of postjunctional folds. This review summarizes these recent results about AChR clustering, which indicate that early clustering can occur in the absence of nerves, that AChRs play an active role in the clustering process and that partly different mechanisms direct formation versus stabilization of AChR clusters. Received 10 April 2002; received after revision 4 June 2002; accepted 10 June 2002     

Neuropeptide Y: the universal soldier

The peptidic neurotransmitter neuropeptide Y (NPY) has received great attention because it has been implicated in the regulation of several organ systems. In particular, NPY is involved in the regulatory loops that control food intake in the hypothalamus and appears also to be important for regulating the activity of neuroendocrine axes under poor metabolic conditions. Furthermore, NPY exerts vasoconstrictive action on the vasculature and potentiates the actions of many other vasoconstrictors. In addition, it was demonstrated to have trophic properties and could therefore contribute to cardiovascular remodeling. These various effects plus a number of others make NPY an attractive target for the potential treatment of human diseases, such as obesity, metabolic disorders, hypertension and heart failure. Received 17 July 2002; received after revision 7 November 2002; accepted 29 November 2002 RID="*" ID="*"Corresponding author.     

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.     

Arginine-rich cell penetrating peptides: from endosomal uptake to nuclear delivery

Delivery of macromolecules into living cells by arginine-rich cell penetrating peptides (AR-CPPs) is an important new avenue for the development of novel therapeutic strategies. However, to date the mechanism of this delivery remains elusive. Recent data implicate endocytosis in the internalization of AR-CPPs and their macromolecular cargo and also indicate limited delivery of macromolecules into the cell cytoplasm and nucleus. Different types of endocytosis – clathrin-dependent endocytosis, raft/caveolin-dependent endocytosis and macropinocytosis – are all implicated in the uptake of AR-CPPs and their cargo into different cells. Cationic AR-CPPs dramatically increase uptake of conjugated molecules through efficient binding to surface proteoglycans. Whether this increase in binding can assure delivery of a sufficient amount of functionally active macromolecules into the cytoplasm and nucleus or whether there is a specific mechanism by which AR-CPPs facilitate the escape of conjugated cargo from endosomes remains to be understood. Received 30 June 2005; received after revision 9 August 2005; accepted 30 August 2005     

Expression of nerve growth factor-like polypeptides and immunoreactivity related to the two types of neurotrophin receptors in earthworm tissues

Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress. Received 12 November 2001; received after revision 8 January 2002; accepted 8 January 2002     

Nanocarriers’ entry into the cell: relevance to drug delivery

Nanocarriers offer unique possibilities to overcome cellular barriers in order to improve the delivery of various drugs and drug candidates, including the promising therapeutic biomacromolecules (i.e., nucleic acids, proteins). There are various mechanisms of nanocarrier cell internalization that are dramatically influenced by nanoparticles’ physicochemical properties. Depending on the cellular uptake and intracellular trafficking, different pharmacological applications may be considered. This review will discuss these opportunities, starting with the phagocytosis pathway, which, being increasingly well characterized and understood, has allowed several successes in the treatment of certain cancers and infectious diseases. On the other hand, the non-phagocytic pathways encompass various complicated mechanisms, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis, which are more challenging to control for pharmaceutical drug delivery applications. Nevertheless, various strategies are being actively investigated in order to tailor nanocarriers able to deliver anticancer agents, nucleic acids, proteins and peptides for therapeutic applications by these non-phagocytic routes.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Chemotherapy and immunotherapy of malignant glioma: molecular mechanisms and clinical perspectives

Despite the considerable progress in modern tumor therapy, the prognosis for patients with glioblastoma, the most frequent malignant brain tumor, has not been substantially improved. Although cytoreductive surgery and radiotherapy are the mainstays of treatment for malignant glioma at present, novel cytotoxic drugs and immunotherapeutic approaches hold great promise as effective weapons against these malignancies. Thus, great efforts are being made to enhance antitumoral efficacy by combining various cytotoxic agents, by novel routes of drug administration, or by combining anticancer drugs and immune modulators. Immunotherapeutic approaches include cytotoxic cytokines, targeted antibodies, and vaccination strategies. However, the success of most of these experimental therapies is prevented by the marked molecular resistance of glioma cells to diverse cytotoxic agents or by glioma-associated immunosuppression. One promising experimental strategy to target glioma is the employment of death ligands such as CD95 (Fas/Apo1) ligand or Apo2 ligand (TRAIL). Specific proapoptotic approaches may overcome many of the obvious obstacles to a satisfactory management of malignant brain tumors. Received 8 March 1999; received after revision 27 May 1999; accepted 14 June 1999     

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation. Received 4 December 2001; received after revision 28 January 2002; accepted 31 January 2002     

Pathogenesis of Plasmodium falciparum malaria: the roles of parasite adhesion and antigenic variation

Malaria results in up to 2.5 million deaths annually, with young children and pregnant women at greatest risk. The great majority of severe disease is caused by Plasmodium falciparum. A characteristic feature of infection with P. falciparum is the accumulation or sequestration of parasite-infected red blood cells (RBCs) in various organs, such as the brain, lung and placenta, and together with other factors is important in the pathogenesis of severe forms of malaria. Sequestration results from adhesive interactions between parasite-derived proteins expressed on the surface of infected RBCs and a number of host molecules on the surface of endothelial cells, placental cells and uninfected RBCs. Some receptors for parasite adhesion have been implicated in particular malaria syndromes, such as intercellular adhesion molecule 1 in cerebral malaria and chondroitin sulfate A and hyaluronic acid in placental infection. The principal parasite ligand and antigen on the RBC surface, P. falciparum erythrocyte membrane protein 1 encoded by a multigene family termed var, is clonally variant, enabling evasion of specific immune responses. An understanding of these host-parasite interactions in the context of clinical disease and immunity may reveal potential targets to prevent or treat severe forms of malaria. Received 25 June 2001; received after revision 22 August 2001; accepted 24 August 2001     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents

Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth.     

Retinoic acid modulates gap junctional intercellular communication in hepatocytes and hepatoma cells

Gap junctional communication permits the direct exchange of small molecules and ions and has been implicated in tissue homeostasis/metabolite exchange. The lack of gap junctional intercellular communication (GJIC) plays important roles in the promotion and progression of carcinogenesis. In the present study, we demonstrate that treatment of human hepatoma Hep G2 cells with retinoic acid (RA) results in increased amounts and phosphorylation of connexins, their stabilisation in plasma membrane plaques and enhanced GJIC. In cultured fetal hepatocytes, which represent a non-transformed, proliferating and incompletely differentiated liver system, the effects of RA are limited to the establishment of connexin in areas of cell-cell contact and the improvement of GJIC. This suggests that modulation of cell-cell channel communication by RA occurs differently in these two experimental models: while RA is able to revert cell transformation in Hep G2 cells, in fetal hepatocytes it may induce the expression of a more differentiated phenotype. Received 19 June 2002; received after revision 29 July 2002; accepted 8 August 2002 RID="*" ID="*"Corresponding author.     

Amelogenin gene splice products: potential signaling molecules

The amelogenins, the major proteins of the developing tooth enamel matrix, are highly conserved throughout most species studied. The gene structure is similar, with a set of seven exons and intervening introns, and remarkable conservation of particular exon sizes over divergent species. Studies of exon skipping and consequent alternative gene splicing suggest that, in vertebrates, exon definition is crucial. In this mechanism, exon size is important. If too small, an exon can be readily skipped, if too large, internal cryptic splice sites may be utilized. Other factors, such as intron length and specific nucleotide sequences at the splice boundaries also modulate splicing efficiency, but amelogenin gene splicing conforms well to the generalized exon length model. Exons 1, 2 and 7 are not subject to splicing that affects the secreted protein product, but exons 3, 4 and 5 are at the lower boundary of exon size, rendering them, 4 and 5 especially, subject to skipping. On the other hand, exon 6 is very long and has cryptic splicing sites that can be used. In the mouse, nine distinct splice product proteins have been detected. The question now is the functions of these products. The larger forms, those that contain the intact proline-rich, hydrophobic exon 6 domains, are important for enamel mineralization. Recent work suggests that the small proteins resulting from deletion of a major part of amelogenin gene exon 6 via utilization of a cryptic site may have signal transduction functions during tooth development. Furthermore, new work also suggests that odontoblasts transiently express the small amelogenins during the period that epithelial-mesenchymal signaling between preodontoblasts and preameloblasts determines the course of tooth development. The same peptides have been demonstrated to act on non-odontogenic cells and effect their phenotypic expression patterns in vitro, and to induce bone formation in implants in vivo. Received 20 March 2002; received after revision 2 July 2002; accepted 3 July 2002     

Statins: the new aspirin?

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been described as the principal and the most effective class of drug to reduce serum cholesterol levels. Statin therapies have been shown to reduce cardiovascular events, including myocardial infarction, stroke, and death, significantly, by altering vascular atherosclerosis development in patients with or without coronary artery disease symptoms. Extensive use of statins has led to the increase of some undesirable effects that are heavily counterbalanced by the benefits. Indeed, pleiotropic effects extend far beyond cholesterol reduction and involve non-lipid-related mechanisms that modify endothelial functions, immunoinflammatory responses, smooth muscle cell activation, proliferation and migration, atherosclerotic plaque stability, and thrombus formation. In this review, we describe in detail the targets and mechanisms of action of statins. Received 6 June 2002; received after revision 6 September 2002; accepted 6 September 2002 RID="*" ID="*"Corresponding author.