Showing papers in "Journal of Molecular Recognition in 2003"

Long-range and short-range mechanisms of hydrophobic attraction and hydrophilic repulsion in specific and aspecific interactions

Abstract: Among the three different non-covalent forces acting in aqueous media, i.e. Lifshitz-van der Waals (LW), Lewis acid-base (AB) and electrical double layer (EL) forces, the AB forces or electron-acceptor/electron-donor interactions are quantitatively by far the predominant ones. A subset of the AB forces acting in water causes the hydrophobic effect, which is the attraction caused by the hydrogen-bonding (AB) free energy of cohesion between the water molecules which surround all apolar as well as polar molecules and particles when they are immersed in water. As the polar energy of cohesion among water molecules is an innate property of water, the hydrophobic attraction (due to the hydrophobic effect) is unavoidably always present in aqueous media and has a value of DeltaG(hydrophobic) = -102 mJ/m(2), at 20 degrees C, being equal to the AB free energy of cohesion between the water molecules at that temperature. The strong underlying hydrophobic attraction due to this effect can, however, be surmounted by very hydrophilic molecules and particles that attract water molecules more strongly than the free energy of attraction of these molecules or particles for one another, plus the hydrogen-bonding free energy of cohesion between the water molecules, thus resulting in a net non-electrical double layer repulsion. Each of the three non-covalent forces, LW, AB or EL, any of which can be independently attractive or repulsive, decays, dependent on the circumstances, as a function of distance according to different rules. These rules, following an extended DLVO (XDLVO) approach, are given, as well as the measurement methods for the LW, AB and EL surface thermodynamic properties, determined at "contact". The implications of the resulting hydrophobic attractive and hydrophilic repulsive free energies, as a function of distance, are discussed with respect to specific and aspecific interactions in biological systems. The discussion furnishes a description of the manner by which shorter-range specific attractions can surmount the usually much stronger long-range aspecific repulsion, and ends with examples of in vitro and in vivo effects of hydrophilization of biopolymers, particles or surfaces by linkage with polyethylene oxide (PEO; also called polyethylene glycol, PEG).

Solid supports for microarray immunoassays

Abstract: Stimulated by the achievements of the first phase in genomics and the resulting need of assigning functions to the acquired sequence information, novel formats of immunoassays are being developed for high-throughput multi-analyte studies. In principle, they are similar in nature to the microarray assays already established at the level of nucleic acids. However, the biochemical diversity and the sheer number of proteins are such that an equivalent analysis is much more complex and thus difficult to accomplish. The wide range of protein concentration complicates matters further. Performing microarray immunoassays already represents a challenge at the level of preparing a working chip surface. Arrays have been produced on filter supports, in microtiter plate wells and on glass slides, the last two usually coated with one-, two- or three-dimensionally structured surface modifications. The usefulness and suitability of all these support media for the construction and application of antibody microarrays are reviewed in this manuscript in terms of the different kinds of immunoassay and the various detection procedures. Additionally, the employment of microarrays containing alternative sensor molecules is discussed in this context. The sensitivity of microspot immunoassays predicted by the current analyte theory is not yet a reality, indicating the extent of both the technology's potential and the size of the task still ahead.

A brief introduction to cell‐penetrating peptides

Abstract: Cell membranes act as protective walls to exclude most molecules that are not actively imported by living cells. This is an efficient way for a cell to prevent uncontrolled influx or efflux of solutes, which otherwise would be harmful to it. Only compounds within a narrow range of molecular size, polarity and net charge are able to diffuse effectively through cell membranes. In order to overcome this barrier for effective delivery of membrane-impermeable molecules, several chemical and physical methods have been developed. These methods, e.g. electroporation, and more recent methods as cationic lipids/liposomes, have been shown to be effective for delivering hydrophobic macromolecules. The drawbacks of these harsh methods are, primarily, the unwanted cellular effects exerted by them, and, secondly, their limitation to in vitro applications. The last decade's discovery of cell-penetrating peptides translocating themselves across cell membranes of various cell lines, along with a cargo 100-fold their own size, via a seemingly energy-independent process, opens up the possibility for efficient delivery of DNA, antisense peptide nucleic acids, oligonucleotides, proteins and small molecules into cells both in vitro and in vivo.

Survey of the year 2005: literature on applications of isothermal titration calorimetry.

Abstract: Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naive, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding.

BEPITOPE: predicting the location of continuous epitopes and patterns in proteins.

Abstract: In growing need of obtaining highly specific monoclonal antibodies against novel proteins, we developed new functions implemented in the program BEPITOPE to predict continuous protein epitopes. This program not only can compute, combine, display and print prediction profiles, but also provides a list of suggested linear peptides to be synthesized. Novel facilities incorporated in BEPITOPE include the treatment of a whole genome, the search for a user-defined pattern, and the combination of prediction to pattern profiles. This latter approach is useful to remove unwanted predictions such as those including glycosylation sites.

Cellular utake of the Tat peptide: an endocytosis mechanism following ionic interactions

Abstract: The cellular delivery of various biological compounds has recently been improved by conjugating them to short peptides known as protein transduction domains or cell penetrating peptides. These peptides include Tat, Antennapedia and arginine-rich peptides. The common feature of these peptides is their highly cationic nature. Up to now, the cellular uptake of about 50 different peptides and proteins coupled to Tat or Antennapedia peptides has been reported. The ability to deliver molecules into cells is not limited to peptide moieties, since oligonucleotides, peptide nucleic acids or other low molecular weight entities have been successfully internalized. Moreover, most of these examples have been accompanied by the expected biological response. More surprisingly, the uptake of large structures such as liposomes, phages, nanoparticles or adenoviruses has also been documented. Indeed the mechanism by which these very different entities could enter cells following a putative common pathway appeared more and more intriguing after each new reported example of cellular uptake mediated by these peptides. After a long period of uncertainty regarding the mechanism of entry, data from several groups now argue for an energy-dependent process of entry. The entry of most of these molecules is likely to be inhibited by low temperature incubation or in the presence of various drugs applied to inhibit the energy-dependent pathway of cell entry. Moreover, the binding of the highly cationic Tat peptide to various anionic membrane components probably initiates the first step of the cell internalization process.

Membrane permeability commonly shared among arginine‐rich peptides

Abstract: Delivery of proteins and other macromolecules using membrane-permeable carrier peptides is a recently developed novel technology, which enables us to modulate cellular functions for biological studies with therapeutic potential. One of the most often used carrier peptides is the arginine-rich basic peptide derived from HIV-1 Tat protein [HIV-1 Tat (48-60)]. Using this peptide, efficient intracellular delivery of molecules including proteins, oligonucleic acids and liposomes has been achieved. We have demonstrated that these features were commonly shared among many arginine-rich peptides such as HIV-1 Rev (34-50) and octaarginine. Not only the linear peptides but also branched-chain peptides showed efficient internalization with an optimum number of arginines (approximately eight residues). The structural and mechanistic features of the translocation of these membrane-permeable arginine-rich peptides are reviewed.

Membrane translocation of penetratin and its derivatives in different cell lines.

Abstract: The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized:The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. (1)H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane.

A survey of the year 2002 commercial optical biosensor literature.

Abstract: We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.

Recombinant antibodies with MHC-restricted, peptide-specific, T-cell receptor-like specificity: new tools to study antigen presentation and TCR-peptide-MHC interactions.

Abstract: The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of antigen-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells or detect antigen-presenting cells (APCs) in tissues. Here we describe a new approach that enables study of human class I peptide-MHC ligand-presentation as well as TCR-peptide-MHC interactions. Such studies are facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant antibodies directed toward a large variety of tumor-associated as well as viral T-cell epitope peptides. Using a large human antibody phage display library, a large panel of recombinant antibodies that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner was isolated. These antibodies were used to directly visualize the specific MHC-peptide complex on tumor cells, antigen-presenting cells or virus-infected cells by flow cytometry. They enabled direct quantitation of the number of MHC-peptide complexes as well as in situ detection of the complex on the surface of APCs after naturally occurring active intracellular processing of the cognate antigen. These studies will enable also the development of a new class of targeting molecules to deliver drugs or toxins to tumor or virus-infected cells. Thus, we demonstrate our ability to transform the unique fine specificity but low intrinsic affinity of TCRs into high-affinity soluble antibody molecules endowed with a TCR-like specificity toward human tumor or viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

Destination 'lysosome': a target organelle for tumour cell killing?

Abstract: Lysosomes and lysosome-related organelles constitute a system of acid compartments that interconnect the inside of the cell with the extracellular environment via endocytosis, phagocytosis and exocytosis. In recent decades it has been recognized that lysosomes are not just wastebaskets for disposal of unused cellular constituents, but that they are involved in several cellular processes such as post-translational maturation of proteins, degradation of receptors and extracellular release of active enzymes. By complementing the autophagic process, lysosomes actively contribute to the maintenance of cellular homeostasis. Proteolysis by lysosomal cathepsins has been shown to mediate the death signal of cytotoxic drugs and cytokines, as well as the activation of pro-survival factors. Secreted lysosomal cathepsins have been shown to degrade protein components of the extracellular matrix, thus contributing actively to its re-modelling in physiological and pathological processes. The malfunction of lysosomes can, therefore, impact on cell behaviour and fate. Here we review the role of lysosomal hydrolases in several aspects of the malignant phenotype including loss of cell growth control, altered regulation of cell death, acquisition of chemoresistance and of metastatic potential. Based on these observations, the lysosome is proposed as a potential target organelle for the chemotherapy of tumours. We will also present some recent data concerning the technologies for delivering chemotherapeutic drugs to the endosomal-lysosomal compartment and the strategies to improve their efficacy.

Immunomodulation by the copolymer glatiramer acetate.

Abstract: Glatiramer acetate (GA; Copaxone®, also known as Copolymer 1 or Cop-1), a copolymer of amino acids, is very effective in the suppression of experimental autoimmune encephalitis (EAE), the animal model for multiple sclerosis (MS), in various species including primates. The immunological cross-reaction between the myelin basic protein and GA serves as the basis for the suppressive activity of GA in EAE, by the induction of antigen-specific suppressor cells. The mode of action of GA is by initial strong promiscuous binding to major histocompatibility complex class II molecules and competition with MBP and other myelin proteins for such binding and presentation to T cells. Suppressor T cells induced by GA are of the Th2 type, migrate to the brain and lead to in situ bystander suppression. Clinical trials with GA, both phase II and phase III, were performed in relapsing–remitting MS (RRMS) patients, and demonstrated efficacy in reducing the relapse rate, decreasing MRI-assessed disease activity and burden and slowing progression of disability. GA is generally well tolerated and is not associated with influenza-like symptoms and formation of neutralizing antibodies seen with β-interferons. It exerts its suppressive effect primarily by immunomodulation, and has recently shown ameliorating effect in a few additional autoimmune disorders as well as in graft rejection. At present GA is considered a valuable first-line treatment option for patients with RRMS. Copyright © 2003 John Wiley & Sons, Ltd.

An improved model of association for VH–VL immunoglobulin domains: Asymmetries between VH and VL in the packing of some interface residues

Abstract: The antibody-binding site is formed as a result of the association between VH and VL domains. Several studies have shown that this association plays an important role in the mechanism of antigen-antibody interaction (Stanfield et al. Structure 1: 83-93, 1993). Considering this, we propose that variations in the VH-VL association are part of the diversification strategy of the antibody repertoires. Previously, a model of association for VH-VL domains based on geometrical characteristics of the packing at the interface was developed by Chothia et al. (J. Mol. Biol. 186: 61-663, 1985). This model includes a common association form for antibodies and a three-layer structure for the interface. In the present work, a complementary model is introduced to account for the general geometrical restrictions of the VH-VL interface, and particular arrangements related to the chemical properties or the side-chain orientations of participating residues. Groups of residues assume common side-chain orientations, which are apparently related to particular functions of different interface zones. Analyses of amino acid usage and network are in agreement with the side-chain orientation patterns. Based on these observations, a three-zone model has evolved to illuminate geometrical and functional restrictions acting over the VH-VL interface. Additionally, this study has revealed the asymmetrical relationships between VH and VL residues important for the association of the two domains.

Measurement of dissociation constants of inhibitors binding to Src SH2 domain protein by non-covalent electrospray ionization mass spectrometry.

Abstract: A mass spectrometric protocol for identifying ligands with a wide range of affinities (3–101 µM) and quantitative spectral analysis for non-covalent interactions have been developed using Src SH2 as a target. Dissociation constants of five compounds, three with a phospho moiety, one with a sulphonic acid group and one with carboxylic acid groups only, were determined using one-ligand one-binding-site, two-ligands one-binding site and one-ligand two-binding-sites models. The Kd values determined by ESI-MS of the three compounds containing the phospho moiety (3.2–7.9 µM) were comparable to those obtained from a solution equilibrium fluorescence polarization assay. The compound with a sulphonate group is a much weaker binding ligand (Kd = 101 µM by ESI, ≫300 µM by FP) towards the Src SH2 protein. Two complexes with different stoichiometric ratios 1:1 and 2:1 (ligand–protein) were observed by ESI-MS for the ligand GIXXX630X. Analysis of binding isotherms indicated the presence of two binding sites for the ligand with Kd values of 9.3 and 193 µM. These data confirmed that, for these polar compounds, non-covalent ESI-MS can measure affinity which very closely reflects the affinity measured under true solution equilibrium conditions. ESI-MS has several key advantages over many solution methods: it can identify the existence of and measure the affinity of complexes other than simple 1:1 ligand–enzyme complexes. Moreover, ESI-MS competition experiments can be readily performed to yield data on whether two ligands bind simultaneously or competitively at the same time as measuring the affinity of the ligand. Copyright © 2003 John Wiley & Sons, Ltd.

Synthesis, characterization and immunochemical evaluation of cephalosporin antigenic determinants.

Abstract: Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms involved in the specific recognition and binding of IgE molecules to these antigenic determinants. We further resolve the relationship between structure and activity of proposed antigenic chemicals, including the rational design and synthesis of these haptenic structures. Comparative RAST inhibition studies of the synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin antibiotics. Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the beta-lactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course.

Targeting dendritic cells for priming cellular immune responses

Abstract: The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immune responses against all classes of pathogens and also against tumors is well established. Their unique potential both to maintain self-tolerance and to initiate protective immune responses against foreign and/or dangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC lining antigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compounds including proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carrying such structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with high endocytic capacity, which provides potential targets for delivering designed molecules. The best route for targeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, which reside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolic products, which induce mobilization of cells to draining lymph nodes where they act as highly potent professional antigen presenting cells. This is brought about by the ability to present their accumulated intracellular content for both CD4+ helper (Th) and CD8+ cytotoxic/cytolytic T lymphocytes (Tc/CTL). Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cell surface in the context of major histocompatibility complex encoded class I and II molecules for presentation to Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigen presentation but also on other features of DC. Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting and T cell-activating functions of DC at their immature and activated differentiation states. These biological activities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interaction of DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that monocyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptive immune responses against pathogens and also against tumors.

The Molecular Basis of Celiac Disease

Abstract: Celiac disease is caused by inflammatory, gluten specific T cell responses in the small intestine. Invariably such responses are HLA-DQ2 or HLA-DQ8 restricted, providing an explanation for the strong association between celiac disease and these HLA-class II alleles. It is now clear that some native gluten sequences can bind to HLA-DQ2/8 and induce T cell responses. In addition, modification of gluten peptides by the enzyme tissue transglutaminase results in high affinity HLA-DQ2/8 binding peptides that can induce T cell responses. Thus, gluten molecules contain a large number of immunogenic peptides and this is likely to play an important role in the breaking of oral tolerance to gluten.

RNA aptamers specific for bovine thrombin.

Abstract: Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a Kd of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished. Copyright © 2003 John Wiley & Sons, Ltd. Abbreviations used: SELEX systematic evolution of ligands by exponential enrichment

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays.

Abstract: The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.

Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting.

Abstract: Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.

Conformational restrictions in the active site of unliganded human caspase-3.

Abstract: Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.

Novel daunorubicin-carrier peptide conjugates derived from human calcitonin segments.

Abstract: Severe and often therapy-limiting side effects are a major obstacle in cancer chemotherapy. New delivery concepts reducing systemic side effects are needed in order to optimize anticancer therapies. Several approaches have been followed, most of them concentrating on macromolecular carriers like liposomes, monoclonal antibodies, serum proteins or polyethylene glycol. We present here a novel type of anthracycline conjugate, using a small carrier peptide derived from the peptide hormone human calcitonin (hCT). The carrier peptide hCT(9-32) has so far been shown to be capable of transporting fluorophores or proteins across cellular membranes. Two different carrier peptide-daunorubicin conjugates were prepared, one with an acid-stable amide bond, the second with an acid-labile hydrazone bond. In vitro studies with daunorubicin linked to the carrier peptide via an acid-labile hydrazone bond demonstrated comparable cytotoxicity to daunorubicin in various daunorubicin sensitive cell lines (neuroblastoma cell lines SK-N-MC and SMS-KAN; HEK 293 T cells). In addition, fluorescence microscopy provided further insight into the mechanism of uptake of the carrier peptide hCT(9-32), indicating that endosomal compartments with reduced pH are involved in the intracellular release of daunorubicin.

Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry.

Abstract: The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.

Radiolabeled α‐melanocyte‐stimulating hormone analogs for receptor‐mediated targeting of melanoma: from tritium to indium

Abstract: Following the first synthesis of tritiated a-melanocyte-stimulating hormone (a-MSH, α-melanotropin) in 1974 by Medzihradszky et al., several α-MSH analogs were designed containing between 2 and 12 tritium atoms, the latter of which displayed a specific radioactivity of 12.21 GBq/μmol (330 Ci/mmol). Similarly, radioiodinated α-MSH analogs of high purity, full biological activity and a specific radioactivity of approximatly 140 GBq/μmol were obtained. Although tritiated and radioiodinated α-MSH became indispensable tools as tracer molecules for numerous in vitro and in vivo studies, above all for receptor identification and characterization as well as for structure-activity studies, they did not fulfill the criteria required for therapeutic in vivo targeting of metastatic melanoma. Therefore, we recently developed α-MSH analogs containing the universal metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in different positions of the molecule. As DOTA can equally well incorporate diagnostic (e.g. 1 1 1 In, 6 7 , 6 8 Ga) and therapeutic (e.g. 9 0 Y, 6 7 Cu) radionuclides, DOTA-MSH compounds may serve for both melanoma scintigraphy and therapy. The analog DOTA-[βAla 3 , Nle 4 , Asp, D-Phe 7 , Lys 1 0 ]-α-MSH 3 - 1 0 (DOTA-MSH O C T ), which contains the metal chelator at its N-terminal end, displayed good in vitro MC1R affinity (IC 5 0 o 9.21 nM). In vivo, [ 1 1 1 In]DOTA-MSH O C T exhibited a favorable biodistribution profile after injection in B16-F1 tumor-bearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4h after injection of [ 1 1 1 In]DOTA-MSH O C T . A comparative study of [ 1 1 1 In]DOTA-MSH O C T with [ 1 1 1 In]DOTA-[Nle 4 , D-Phe 7 ]-α-MSH ([ 1 1 1 In]DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH O C T peptide, particularly with respect to the amount of radioactivity taken up by non-malignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [ 1 1 1 In]DOTA-MSH O C T specifically targets melanoma metastases and represents a lead compound for the development of therapeutic DOTA-MSH analogs.

Multiple-point adsorption of terbium ions by lead ion templated thermosensitive gel: elucidating recognition of conformation in gel by terbium probe.

Abstract: Lead ion templated thermosensitive heteropolymer gel which has recognition ability of methacrylate pairs has been synthesized and characterized. The gel consists of a main monomer component, N-isopropylacrylamide (NIPA), responsible for volume phase transition, methacrylic acid (MAA) moieties imprinted as pairs to adsorb terbium ions and cross-links. An imprinting technique was applied using lead ion complex with methacrylate ligands in dioxane media. After gel was obtained, lead ions were removed by washing and the imprinted gel showed strong binding ability to terbium ions, comparable with that of the non-imprinted gel prepared without lead ions. It was found that the Tb3+ fluorescence intensity was considerably increased upon binding this ion to both imprinted and non-imprinted gels, but the largest enhancement of fluorescence intensity was observed when Tb3+ was bound to imprinted gel in shrunken state. This is because of the decrease of coordinated water molecules on Tb3+ and the strong binding of this ion to methacrylate pairs which are encoded within the weakly cross-linked network of imprinted gel. Copyright © 2003 John Wiley & Sons, Ltd.

Analysis of the pH-dependencies of the association and dissociation kinetics of HIV-1 protease inhibitors.

Abstract: The kinetic constants for the interactions between HIV-1 protease and a selection of inhibitors were determined at different pH-values using a biosensor based interaction assay. Since this technique does not involve a substrate, it was possible to determine the pH-dependencies of the association and dissociation rates of an inhibitor, without the complication of a pH-dependent enzyme-substrate/product equilibrium. The importance of these interactions was evaluated by correlating the free energy changes upon association and dissociation of inhibitors with the predicted change in electrostatic properties of the interacting groups as a result of altered pH. It was found that the kinetic parameters varied with pH in a unique manner for all inhibitors, demonstrating that the kinetic features were associated with the specific structure of each inhibitor. Association and dissociation had different pH-profiles, indicating that the two processes proceeded by different pathways/mechanisms. The energy barrier for dissociation of the enzyme-indinavir complex increased with pH from 4.1 to 7.4, while it was generally reduced for the other inhibitors as the pH was increased from 5.1 to 7.4. The pH-dependent interactions involved in the recognition/binding of inhibitors and in the stabilization of the complex were identified by analysing three-dimensional structures of enzyme-inhibitor complexes. The interaction between the pyridine nitrogen of indinavir with Arg-8 was hypothesized to be responsible for the unique pH-dependency of indinavir. The analysis revealed features of interactions that are significant for understanding enzyme function and for optimization of new drug leads. It also highlighted the importance of environmental conditions on interactions.

Therapeutic approaches to superantigen-based diseases: a review.

Abstract: Superantigens secreted by the bacterial pathogen Staphyloccocus aureus are extremely potent toxins that overstimulate the host immune system by binding to the MHC class II and T cell receptors and activating a large population of T cells. Superantigen infection has been shown to be the causative agents in acute diseases, food poisoning and toxic shock syndrome, and in more chronic conditions such as inflammatory skin diseases. In addition to the toll on public health, S. aureus superantigens also represent a potential biothreat to our national security. To address these risks, a number of different therapeutic strategies have been developed that target different aspects of the pathogenic mechanism of S. aureus and superantigen infection. These therapies, which encompass strategies as diverse as production of neutralizing antibodies, inhibitory peptide/receptor design and blockage of superantigen gene transcription, are being tested for treatment of established S. aureus infections in pre- and post-exposure scenarios. In this review, we will describe these different strategies and their efficacies in inhibition of superantigen-induced effects in the host, and present the future outlook for successfully producing therapies for superantigen-based disease.

Human CD30: structural implications from epitope mapping and modeling studies.

Abstract: The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.

Structure‐based discovery of a new affinity ligand to pancreatic α‐amylase

Abstract: A ligand useful for affinity capture of porcine pancreatic α-amylase was found by virtual screening of the commercially available compound data base MDL® Available Chemicals Directory. Hits from the virtual screening were investigated for binding by nuclear magnetic resonance (NMR) and surface plasmon resonance. Selected compounds were tested for inhibition of the enzyme using a NMR-based assay. One of the binders found was covalently coupled to a chromatographic resin and a column, packed with this resin, could retain α-amylase, which subsequently was eluted by introduction of the known inhibitor acarbose to the elution buffer. Copyright © 2003 John Wiley & Sons, Ltd.

Molecular tools for targeted imaging and therapy of cancer.

Abstract: Here we review an approach to the design and production of antibody/ligand pairs for use in cell targeting procedures, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This methodology is applicable to other biological binding pairs.