Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

G-protein coupled receptor assays: to measure affinity or efficacy that is the question

Cell-based assays have always played an important role in the pharmaceutical industry, providing information about the functional effects of compounds. These functional assays have traditionally accompanied facile biochemical high throughput screening programmes, being applied as secondary assays in the later stages of lead development. However, with the disappointing reality that there is not likely to be a plethora of novel, druggable targets in the post-genomic era, the role of cell-based assays in drug discovery is beginning to change. Competition to develop the "best" agents for well established targets and find more effective ways of identifying "novel" agents is driving the industry towards a "quality" versus "quantity" approach. Advances in genetic engineering, automation compatible functional assay technologies and the introduction of more sophisticated robotic systems, have facilitated the application of cell-based assays to primary screening. However, despite some apparent success to move these assays into the routine "toolbox" for high throughput screening, certain preconceptions and concerns about cell-based assays persist and the subject remains a topic of much debate. Here we use examples from the screening portfolio at Pfizer, Sandwich, to discuss the practical and theoretical considerations of employing cell-based assays in HTS with a focus on G-protein coupled receptors.     

Micro-patterned porous substrates for cell-based assays

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.     

Porphyrin Metalation at MgO Surfaces: A Spectroscopic and Quantum Mechanical Study on Complementary Model Systems

We show that both single‐crystalline and nanostructured MgO surfaces convert free‐base tetraphenyl porphyrin (2HTPP) into magnesium tetraphenyl porphyrin (MgTPP) at room temperature. The reaction can be viewed as an ion exchange between the two aminic protons of the 2HTPP molecule with a Mg²⁺ ion from the surface. The driving force for the reaction is the strong stability of the formed hydroxyl groups along the steps and at defects on the MgO surface. We have used an integrated characterization approach that includes UV/Vis diffuse reflectance measurements on nanostructured powders, X‐ray photoelectron spectroscopic investigation of atomically clean MgO(100) single‐crystalline thin films, and density functional theory (DFT) calculations on model systems. The DFT calculations demonstrate that MgTPP formation is strongly exothermic at the corners, edges and steps, but slightly endothermic on terrace sites. This agrees well with the UV/Vis diffuse reflectance, which upon adsorption of 2HTPP shows a decrease in the absorption band associated with corner and edge sites on MgO nanocube powders.     

Design, synthesis, and characterization of a potent, nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP

XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We report the design, synthesis, and characterization of a nonpeptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000 times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultrapotent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in the HL-60 leukemia cell line. The potency of bivalent SM-164 in binding, functional, and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics.     

Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials

The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.     

Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X(L)

Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.     

Array-based capture, distribution, counting and multiplexed assaying of beads on a centrifugal microfluidic platform

We present a novel centrifugal microfluidic platform for the highly efficient manipulation and analysis of particles for applications in bead-based assays. The platform uses an array of geometrical V-cup barriers to trap particles using stopped-flow sedimentation under highly reproducible hydrodynamic conditions. The impact parameters governing the occupancy distribution and capture efficiency of the arrayed traps are investigated. The unique, nearly 100% capture efficiency paired with the capability to establish sharply peaked, single occupancy distributions enables a novel, digital readout mode for color-multiplexed, particle-based assays with low-complexity instrumentation. The presented technology marks an essential step towards a versatile platform for the integration of bead- and cell-based biological assays.     

Intein-mediated biotinylation of proteins and its application in a protein microarray

We report here the first example using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. With this novel array, proteins are site-specifically immobilized on the glass surface and are able to retain their native activity. The advantage of the avidin/biotin linkage over his-tag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which makes this new protein array compatible with most biological assays.     

High content screening for G protein-coupled receptors using cell-based protein translocation assays

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.     

A renewable, chemoselective, and quantitative ligand density microarray for the study of biospecific interactions

Novel renewable microarray technology has been developed to immobilize and release carbohydrates and proteins from self-assembled monolayers (SAMs) of electroactive quinone-terminated alkanethiolates on gold surfaces. This method may be applied to a variety of research fields for use in biosensor technology and the generation of renewable and tailored microarrays for biospecific cell-based assays.     

PPI Modulators of E6 as Potential Targeted Therapeutics for Cervical Cancer: Progress and Challenges in Targeting E6

Advanced cervical cancer is primarily managed using cytotoxic therapies, despite evidence of limited efficacy and known toxicity. There is a current lack of alternative therapeutics to treat the disease more effectively. As such, there have been more research endeavors to develop targeted therapies directed at oncogenic host cellular targets over the past 4 decades, but thus far, only marginal gains in survival have been realized. The E6 oncoprotein, a protein of human papillomavirus origin that functionally inactivates various cellular antitumor proteins through protein–protein interactions (PPIs), represents an alternative target and intriguing opportunity to identify novel and potentially effective therapies to treat cervical cancer. Published research has reported a number of peptide and small-molecule modulators targeting the PPIs of E6 in various cell-based models. However, the reported compounds have rarely been well characterized in animal or human subjects. This indicates that while notable progress has been made in targeting E6, more extensive research is needed to accelerate the optimization of leads. In this review, we summarize the current knowledge and understanding of specific E6 PPI inhibition, the progress and challenges being faced, and potential approaches that can be utilized to identify novel and potent PPI inhibitors for cervical cancer treatment.     

Cell characterization using a protein-functionalized pore

We demonstrate a highly-sensitive and label-free method for characterizing cells based on cell-surface receptors. The method involves measuring a current pulse generated when an individual cell passes through an artificial pore. When the pore is functionalized with proteins, specific interactions between a cell-surface marker and the functionalized proteins retard the cell, thus leading to an increased pulse duration that indicates the presence of that specific biomarker. For proof-of-principle, we successfully screened murine erythroleukemia cells based on their CD34 surface marker in both a single and mixed population of cells. Further, we developed a unified constrained statistical model for estimating the ratios of cells in a mixed population. Finally, we demonstrated our ability to screen a small number of cells (hundreds or less) with high accuracy and sensitivity. Overall, our pore-based method is broadly applicable and, in the future, could provide a full range of in vitro cell-based assays.     

Oscillatory molecular driving force for protein folding at high concentration: a molecular simulation

This paper presents a Langevin dynamics simulation that suggests a novel way to fold protein at high concentration, a fundamental issue in neurodegenerative diseases in vivo and the production of recombinant proteins in vitro. The simulation indicates that the folding of a coarse-grained beta-barrel protein at high concentration follows the "collapse-rearrangement" mechanism but it yields products of various forms, including single proteins in the native, misfolded, and uncollapsed forms and protein aggregates. Misfolded and uncollapased proteins are the "nucleus" of the aggregates that also encapsulate some correctly folded proteins (native proteins). An optimum hydrophobic interaction strength (epsilon*(p)) between the hydrophobic beads of the model protein, which results from a compromise between the kinetics of collapse and rearrangement, is identified for use in increasing the rate of folding over aggregating. Increased protein concentration hinders the structural transitions in both collapse and rearrangement and thus favors aggregation. A new method for protein folding at high concentration is proposed, which uses an oscillatory molecular driving force (epsilon*(p)) to promote the dissociation of aggregates in the low epsilon*(p) regime while promoting folding at a high epsilon*(p). The advantage of this method in enhancing protein folding while depressing aggregation is illustrated by a comparison with the methods based on direct dilution or applying a denaturant gradient.     

Nanoparticles,Proteins, and Nucleic Acids: Biotechnology Meets Materials Science

Based on fundamental chemistry, biotechnology and materials science have developed over the past three decades into today's powerful disciplines which allow the engineering of advanced technical devices and the industrial production of active substances for pharmaceutical and biomedical applications. This review is focused on current approaches emerging at the intersection of materials research, nanosciences, and molecular biotechnology. This novel and highly interdisciplinary field of chemistry is closely associated with both the physical and chemical properties of organic and inorganic nanoparticles, as well as to the various aspects of molecular cloning, recombinant DNA and protein technology, and immunology. Evolutionary optimized biomolecules such as nucleic acids, proteins, and supramolecular complexes of these components, are utilized in the production of nanostructured and mesoscopic architectures from organic and inorganic materials. The highly developed instruments and techniques of today's materials research are used for basic and applied studies of fundamental biological processes.     

Dioxin analysis in feed: cell-based assay versus mass spectrometry method

In the determination of contaminants (dioxins, polychlorinated biphenyls, polyaromatic hydrocarbons), cell-based assays are useful methods for screening purposes: they are mainly characterized by high sample throughput and lower costs than the Mass Spectrometry (MS)-based methods. Although cell-based assays can be sensitive enough for the determination of dioxins and related substances in agreement with the presently tolerable limits in food and feed (Regulation No. 2375/2001/EC and Directive 2003/57/EC respectively), their lack of specificity make their use rather questionable in control laboratories. In this paper, we present and compare results obtained from the analysis of a limited number of feed samples by both gas chromatography-high resolution mass spectrometry (GC-HRMS) and cell-based assay (DR-CALUX: dioxin responsive-chemically activated luciferase gene expression) methods. The DR-CALUX screening led to less than 10% false non-compliant and no false compliant results. In addition, there is a good correlation between GC-HRMS and DR-CALUX data. However, these preliminary results have to be confirmed on a larger number of samples to demonstrate that total toxic equivalent (TEQ), including dioxins, furans and dioxin-like polychlorobiphenyls (PCBs) can be monitored in feed and food with a cell-based assay. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

A cell-based screening method for specifically detecting kinase activity

No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.     

Arsenic trioxide targets Hsp60, triggering degradation of p53 and survivin

The mechanisms of action of arsenic trioxide (ATO), a clinically used drug for the treatment of acute promyelocytic leukemia (APL), have been actively studied mainly through characterization of individual putative protein targets. There appear to be no studies at a system level. Herein, we integrate metalloproteomics through a newly developed organoarsenic probe, As-AC (C₂₀H₁₇AsN₄O₃S₂) with quantitative proteomics, allowing 37 arsenic binding and 250 arsenic regulated proteins to be identified in NB4, a human APL cell line. Bioinformatics analysis reveals that ATO disrupts multiple physiological processes, in particular, chaperone-related protein folding and cellular response to stress. Furthermore, we discover heat shock protein 60 (Hsp60) as a vital target of ATO. Through biophysical and cell-based assays, we demonstrate that ATO binds to Hsp60, leading to abolishment of Hsp60 refolding capability. Significantly, the binding of ATO to Hsp60 disrupts the formation of Hsp60-p53 and Hsp60-survivin complexes, resulting in degradation of p53 and survivin. This study provides significant insights into the mechanism of action of ATO at a systemic perspective, and serves as guidance for the rational design of metal-based anticancer drugs.

A highly selective organoarsenic fluorescent probe As-AC and quantitative proteomics were employed to track arsenic-binding and regulating proteins in live leukemia cells. Hsp60 was validated as a new target of ATO.     

Exploring structure-activity relationships of tricyclic farnesyltransferase inhibitors using ECLiPS libraries

The development of structure-activity relationships (SARs) relating to the function of a biological protein is often a long and protracted undertaking when using an iterative medicinal chemistry approach. High throughput screening of ECLiPS (Encoded Combinatorial Libraries on Polymeric Support) libraries can be used to simplify this process. In this paper, we illustrate how a large ECLiPS library of 26,908 compounds, based on a tricyclic core structure, was used to define a multitude of SARs for the oncogenic target, farnesyltransferase (FTase). This library, FT-2, was prepared using a split-and-pool approach in which small molecules are constructed on resin that contains tag/linker constructs to track the synthetic process [1-5] Highly defined SARs were produced from this screen that enhanced our understanding of FTase binding site interactions. The pivotal compounds culled from this library were potent in both cell-free and cell-based FTase assays, selective over the closely related enzyme, geranylgeranyltransferase I (GGTase I), and inhibited the adherent-independent growth of a transformed cell line.