Novel genomic methods for drug discovery and mechanism-based toxicological assessment

Genomics encompasses a range of powerful technologies that can be applied at all levels of gene expression, from transcription to mRNA translation. Collectively, these technologies have great potential for improving drug discovery, both target and molecule recognition, and development. In this article we review the current and potential future status of established and novel genomic methods within drug discovery.

On the mechanism for optomechanical entanglement and its revelation

An intuitive and accurate picture of the occurrence of optomechanical entanglement in a system of great current experimental interest is provided. At the optimal working point for entanglement generation, the interaction of a light field with a micromechanical oscillator is described in terms of simple SU( 2) transformations. This allows the analysis of a recent proposal for the revelation of quantumness in the state of a massive mirror from a clear and experimentally biased viewpoint.

Suggested Binding Mechanism of the HIV-gp120 to its CD4 Receptor

The molecular recognition and attachment of the CD4 molecule and the HIV envelope glycoprotein (gp120) might be described as a consecutive three-step molecular recognition process. 1. (a) Long range interaction: electrostatic pre-orientation, 2. (b) short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation) and 3. (c) internal interaction (induced fit): conformational readjustment of the protein molecules. On the basis of the preliminary investigations (X-ray structures of CD4 and biological studies of CD4 and gp120 point mutants) we described a computational model. This approach consists of empirical calculations as well as ab initio level of quantum chemistry. The conformational analysis of the wild type and mutant CD4 molecules was supported by molecular mechanics and dynamics (Amber force field). The latter analysis involves the application of a novel method, the Amino Acid Conformation Assignment of Proteins (ACAP) software, developed for the notation of secondary protein structures. According to the cardinal role of the electrostatic factors during this interaction, several ab initio investigations were performed for better understanding of the recognition process on submolecular level. Using the above mentioned computational model, we could interpret the basic behaviours and predict some additional features of CD4-gp120 interaction, in spite of the missing gp120 X-ray structure.

Hypercapnic acidosis attenuates pulmonary epithelial wound repair by an NF-kappa B dependent mechanism

Background: Hypercapnic acidosis exerts protective effects in acute lung injury but may also slow cellular repair. These effects may be mediated via inhibition of nuclear factor-kappa B (NF-kappa B), a pivotal transcriptional regulator in inflammation and repair.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

The top-down mechanism for body-mass-abundance scaling

Scaling relationships between mean body masses and abundances of species in multitrophic communities continue to be a subject of intense research and debate. The top-down mechanism explored in this paper explains the frequently observed inverse linear relationship between body mass and abundance (i.e., constant biomass) in terms of a balancing of resource biomasses by behaviorally and evolutionarily adapting foragers, and the evolutionary response of resources to this foraging pressure. The mechanism is tested using an allometric, multitrophic community model with a complex food web structure. It is a statistical model describing the evolutionary and population dynamics of tens to hundreds of species in a uniform way. Particularities of the model are the detailed representation of the evolution and interaction of trophic traits to reproduce topological food web patterns, prey switching behavior modeled after experimental observations, and the evolutionary adaptation of attack rates. Model structure and design are discussed. For model states comparable to natural communities, we find that (1) the body-mass-abundance scaling does not depend on the allometric scaling exponent of physiological rates in the form expected from the energetic equivalence rule or other bottom-up theories; (2) the scaling exponent of abundance as a function of body mass is approximately -1, independent of the allometric exponent for physiological rates assumed; (3) removal of top-down control destroys this pattern, and energetic equivalence is recovered. We conclude that the top-down mechanism is active in the model, and that it is a viable alternative to bottom-up mechanisms for controlling body-mass-abundance relations in natural communities.

FKBPL and Peptide Derivatives: Novel Biological Agents That Inhibit Angiogenesis by a CD44-Dependent Mechanism

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action.

Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status.

Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype.

Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

Derivation of clinically compliant MSCs from CD105+, CD24-differentiated human ESCs

Adult tissue-derived mesenchymal stem cells ( MSCs) have demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues through mechanisms thought to be mediated by either cell replacement or secretion of paracrine factors. Characterized, self- renewing human ESCs could potentially be an invariable source of consistently uniform MSCs for therapeutic applications. Here we describe a clinically relevant and reproducible manner of generating identical batches of hESC- derived MSC ( hESC- MSC) cultures that circumvents exposure to virus, mouse cells, or serum. Trypsinization and propagation of HuES9 or H1 hESCs in feeder- and serum-free selection media generated three polyclonal, karyotypically stable, and phenotypically MSC-like cultures that do not express pluripotency- associated markers but displayed MSC- like surface antigens and gene expression profile. They differentiate into adipocytes, osteocytes, and chondrocytes in vitro. Gene expression and fluorescence- activated cell sorter analysis identified CD105 and CD24 as highly expressed antigens on hESC- MSCs and hESCs, respectively. CD105+, CD24- monoclonal isolates have a typical MSC gene expression profiles and were identical to each other with a highly correlated gene expression profile ( r(2) >.90). We have developed a protocol to reproducibly generate clinically compliant and identical hESC- MSC cultures.