A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration

How proteins bind macrocycles

The potential utility of synthetic macrocycles (MCs) as drugs, particularly against low-druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of MCs for good target protein-binding activity or bioavailability. To address this knowledge gap, we analyze the binding modes of a representative set of MC-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic MC libraries with structural and physicochemical features likely to favor strong binding to protein targets as well as good bioavailability. We additionally provide evidence that large, natural product-derived MCs can bind targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product-inspired synthetic MCs can expand the number of proteins that are druggable by synthetic small molecules.R01 GM094551 - NIGMS NIH HHS; GM064700 - NIGMS NIH HHS; GM094551 - NIGMS NIH HHS; R01 GM064700 - NIGMS NIH HHS; GM094551-01S1 - NIGMS NIH HH

Translational Oncogenomics and Human Cancer Interactome Networks

An overview of translational, human oncogenomics, transcriptomics and cancer interactomic networks is presented together with basic concepts and potential, new applications to Oncology and Integrative Cancer Biology. Novel translational oncogenomics research is rapidly expanding through the application of advanced technology, research findings and computational tools/models to both pharmaceutical and clinical problems. A self-contained presentation is adopted that covers both fundamental concepts and the most recent biomedical, as well as clinical, applications. Sample analyses in recent clinical studies have shown that gene expression data can be employed to distinguish between tumor types as well as to predict outcomes. Potentially important applications of such results are individualized human cancer therapies or, in general, ‘personalized medicine’. Several cancer detection techniques are currently under development both in the direction of improved detection sensitivity and increased time resolution of cellular events, with the limits of single molecule detection and picosecond time resolution already reached. The urgency for the complete mapping of a human cancer interactome with the help of such novel, high-efficiency / low-cost and ultra-sensitive techniques is also pointed out

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

Monoamine Transporter Photoaffinity Ligands Based On Methylphenidate and Citalopram: Rational Design, Chemical Synthesis, and Biochemical Application

Monoamine transporters (MATs) are a family of proteins that include the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Specifically, dysregulation of MAT function is associated with a host of disease states including drug abuse, major depressive disorder, and anxiety. Additionally, several drugs acting as MAT inhibitors are clinically available to treat multiple disorders. However, details regarding the transport inhibition mechanism created by these drugs, as well as their discrete ligand-binding pockets within their target MAT proteins, remains poorly understood. This knowledge gap in turn hinders rational development of novel therapeutics for numerous MAT-associated disorders. The objective of this research dissertation was to develop irreversible chemical probes based on methylphenidate (MP) and citalopram (CIT), two therapeutically significant MAT inhibitors, in order to map their binding sites and poses within their major MAT target protein. The central hypothesis was that MP and CIT could be rationally derivatized, without significant loss in pharmacological activity, to contain a tag moiety and a photoreactive group capable of forming a covalent bond to their target MAT protein, thus allowing application of a Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL) experimental approach. Specifically, BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling in order to map the binding sites and poses of ligands within their target proteins. This central hypothesis was tested by pursuing three specific aims: 1) identification of non-tropane photoprobes based on MP suitable for DAT structure-function studies, 2) identification of photoprobes based on CIT and ( S )-CIT suitable for SERT structure-function studies, and 3) development of a tandem photoaffinity labeling-bioorthogonal conjugation protocol for SERT structure-function studies. In the first aim, MP was structurally modified to contain an aryl azide photoreactive group and a 125 I radioisotope tag. The compounds were then subjected to DAT pharmacological evaluation in order to identify suitable candidates for DAT structure-function studies. In the second aim, CIT and (S )-CIT were structurally modified to contain an aryl azide or benzophenone photoreactive group and 125 I, a terminal alkyne, or an aliphatic azide as a tag. Likewise, these compounds were subjected to SERT pharmacological evaluation in order to identify suitable candidates for SERT structure-function studies. Finally, under the third aim, a tandem photoaffinity labeling-bioorthogonal conjugation protocol was developed to label purified hSERT expressed in HEK-293 cells using a ( S )-CIT-based benzophenone-alkyne clickable photoprobe. Probe-labeled hSERT samples from this protocol are currently being analyzed by high resolution mass spectrometry in order to map the ( S )-CIT-binding site(s) within the hSERT

Methods in protein engineering and screening : from rational design to directed evolution and beyond

Humans have engineered organisms for thousands of years. The domestication of plants and animals are early examples of how humans have changed genotypes of organisms for prized traits that persist today. The genotype to phenotype linkage is often the result of the expression or performance of proteins found within an organism. The modern era of biological engineering has seen the emergence of two fundamentally distinct philosophies on how best to alter the genetic code of an organism or sequence of a particular protein for desired phenotypic outcomes. Rational design and directed evolution are often seen as two competing methodologies working towards the same goal—the engineering of proteins and ultimately organisms. Here, I attempt to bridge the gap between the two engineering strategies and argue that a combination is needed in many cases. Starting with the rationalization of directed evolution experiments using software tools, we begin to understand the predictive power and limitations of rational computational approaches. We then explore the computational resurfacing of an enzyme and discover an unpredicted thermoswitchable phenotype. Next, we rationally design a number of fusion proteins, which allow us to create novel point-of-care diagnostics. We then turn our attention to the directed evolution of a number of DNA polymerases with novel functions. The amalgamation of rational design and directed evolution are then extended to a eukaryotic organism, which enables us to engineer more complex proteins and gives us the ability to create novel drug screening and discovery platforms.Biochemistr

Titanocene anticancer complexes and their binding mode of action to human serum albumin: a computational study

Due to the pivotal role played by human serum albumin (HSA) in the transport and cytotoxicity of titanocene complexes, a docking study has been performed on a selected set of titanocene complexes to aid in the current understanding of the potential mode of action of these titanocenes upon binding HSA. Analysis of the docking results has revealed potential binding at the known drug binding sites in HSA and has provided some explanation for the specificity and subsequent cytotoxicity of these titanocenes. Additionally, a new alternative binding site for these titanocenes has been postulated