Potential Applications In Drug Discovery Research Articles

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine.

Three-dimensional (3-D) cell printing, which can accurately deposit cells, biomaterial scaffolds and growth factors in precisely defined spatial patterns to form biomimetic tissue structures, has emerged as a powerful enabling technology to create live tissue and organ structures for drug discovery and tissue engineering applications. Unlike traditional 3-D printing that uses metals, plastics and polymers as the printing materials, cell printing has to be compatible with living cells and biological matrix. It is also required that the printing process preserves the biological functions of the cells and extracellular matrix, and to mimic the cell-matrix architectures and mechanical properties of the native tissues. Therefore, there are significant challenges in order to translate the technologies of traditional 3-D printing to cell printing, and ultimately achieve functional outcomes in the printed tissues. So it is essential to develop new technologies specially designed for cell printing and in-depth basic research in the bioprinted tissues, such as developing novel biomaterials specifically for cell printing applications, understanding the complex cell-matrix remodeling for the desired mechanical properties and functional outcomes, establishing proper vascular perfusion in bioprinted tissues, etc. In recent years, many exciting research progresses have been made in the 3-D cell printing technology and its application in engineering live tissue constructs. This review paper summarized the current development in 3-D cell printing technologies; focus on the outcomes of the live printed tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions of 3-D cell printing technology are also discussed.

An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back.

The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery.

Three-dimensional bioprinting and tissue fabrication: prospects for drug discovery and regenerative medicine

Bioprinting technology has emerged as a powerful tool for building tissue and organ structures for drug discovery and regenerative medicine applications. In general, bioprint- ing uses a computer-controlled three-dimensional (3D) printing device to accurately deposit cells and biomaterials into precise geometries with the goal of creating anatomically correct biological structures. While traditional 3D printing uses metals, plastics, and polymers as printing materials or ink, bioprinting deals with living cells and biological matrix. Hence, there are significant challenges to make a transition from traditional 3D printing to bioprint - ing, and ultimately achieve functional outcomes in bioprinted tissues. Therefore, it is critical that there is new technology development and in-depth basic research in bioprinted tissues, such as developing novel biomaterials specifically for use in bioprinting and biofabrication techniques, understanding the cell-matrix remodeling for the desired mechanical properties, and functional outcomes, establishing proper vascular perfusion, etc. Currently, there is active research going on bioprinting technology and its potential as a future source for tissue implants. This review paper overviews the current state of the art in bioprinting technology and focuses on the outcomes of the bioprinted tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions for next-generation bioprinting technology are also presented.

A logical model of HIV-1 interactions with the T-cell activation signalling pathway.

Human immunodeficiency virus type 1 (HIV-1) hijacks host cellular processes to replicate within its host. Through interactions with host proteins, it perturbs and interrupts signaling pathways that alter key cellular functions. Although networks of viral-host interactions have been relatively well characterized, the dynamics of the perturbation process is poorly understood. Dynamic models of infection have the potential to provide insights into the HIV-1 host interaction. We employed a logical signal flow network to model the dynamic interactions between HIV-1 proteins and key human signal transduction pathways necessary for activation of CD4+ T lymphocytes. We integrated viral-host interaction and host signal transduction data into a dynamic logical model comprised of 137 nodes (16 HIV-1 and 121 human proteins) and 336 interactions collected from the HIV-1 Human Interaction Database. The model reproduced expected patterns of T-cell activation, co-stimulation and co-inhibition. After simulations, we identified 26 host cell factors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activation or inhibition of viral proteins. Through in silico knockouts, the model identified a further nine host cell factors, including members of the PI3K signalling pathway that are essential to viral replication. Simulation results intersected with the findings of three siRNA gene knockout studies and identified potential drug targets. Our results demonstrate how viral infection causes the cell to lose control of its signalling system. Logical Boolean modelling therefore provides a useful approach for analysing the dynamics of host-viral interactions with potential applications for drug discovery. Supplementary data are available at Bioinformatics online.

DREADD: a chemogenetic GPCR signaling platform.

Recently, we created a family of engineered G protein-coupled receptors (GPCRs) called DREADD (designer receptors exclusively activated by designer drugs) which can precisely control three major GPCR signaling pathways (Gq, Gi, and Gs). DREADD technology has been successfully applied in a variety of in vivo studies to control GPCR signaling, and here we describe recent advances of DREADD technology and discuss its potential application in drug discovery, gene therapy, and tissue engineering.

Targeting bromodomains: epigenetic readers of lysine acetylation.

Lysine acetylation is a key mechanism that regulates chromatin structure; aberrant acetylation levels have been linked to the development of several diseases. Acetyl-lysine modifications create docking sites for bromodomains, which are small interaction modules found on diverse proteins, some of which have a key role in the acetylation-dependent assembly of transcriptional regulator complexes. These complexes can then initiate transcriptional programmes that result in phenotypic changes. The recent discovery of potent and highly specific inhibitors for the BET (bromodomain and extra-terminal) family of bromodomains has stimulated intensive research activity in diverse therapeutic areas, particularly in oncology, where BET proteins regulate the expression of key oncogenes and anti-apoptotic proteins. In addition, targeting BET bromodomains could hold potential for the treatment of inflammation and viral infection. Here, we highlight recent progress in the development of bromodomain inhibitors, and their potential applications in drug discovery.

Assessment of the Hepatic CYP Reductase Null Mouse Model and Its Potential Application in Drug Discovery

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

Ubiquitin ligases in cholesterol metabolism.

To maintain cholesterol homeostasis, the processes of cholesterol metabolism are regulated at multiple levels including transcription, translation, and enzymatic activity. Recently, the regulation of protein stability of some key players in cholesterol metabolism has been characterized. More and more ubiquitin ligases have been identified including gp78, Hrd1, TRC8, TEB4, Fbw7, and inducible degrader of low density lipoprotein receptor. Their working mechanisms and physiological functions are becoming revealed. Here, we summarize the structure, substrates and function of these ubiquitin ligases. Their potential application in drug discovery is also discussed.

Stem Cell Lineage Commitment by Electrical Fields and the Potential Application in Drug Discovery

Stem cells may be applied to improve the efficiency of drug discovery, but more effective protocols are first required to control the differentiation. Recent researches have revealed that physical stimulation is an important avenue for stem cell lineage commitment, such as electrical field stimulation. Here, we review literatures about stem cell differentiation by electrical field stimulation. Various forms of electrical fields with soluble induction factors have shown to produce a synergistic effect in order to enhance the osteogenic commitment. Moreover, electrical field stimulation alone shows marked effects of pre-commitment to cardiomyocyte and neuron. However, the related precise molecular regulatory mechanism is unclear. As cardiomyocyte and neuron are crucial factors in drug development process, electrical field stimulation may be proposed as an effect important for stem cell differentiation, exhibiting a potential application in drug discovery.

Thermodynamic and kinetic specificities of ligand binding

Binding affinity and specificity are crucial for biomolecular recognition. Past studies have focused on binding affinity while the quantification of specificity has remained an elusive challenge. The conventional specificity measures the discrimination of the specific receptor against others for a ligand binding. It is difficult to explore all the possible competing receptors for the ligand. Here, we quantified the thermodynamic intrinsic specificity of discriminating the “native” binding mode against the “non-native” binding modes. Intrinsic specificity is relatively easy to compute since one doesn't need to explore all the other receptors. We found that the thermodynamic intrinsic specificity correlates with the conventional specificity. This validates the statistical equivalence of conventional and intrinsic specificities for receptors at reasonable size. We also computationally quantified the residence time of a ligand on the receptor target as the kinetic specificity. We found that the kinetic specificity correlates with the thermodynamic intrinsic specificity and the binding affinity, suggesting the kinetics and the thermodynamics can be simultaneously optimized for biological activities. With the thermodynamic and kinetic specificities in addition to affinity, we carried out a drug screening test on the target cyclooxygenase-2 (COX-2). We showed that multidimensional (two- and three-dimensional) screening has higher capability than affinity alone to discriminate the drug target (COX-2) from the competitive receptor (COX-1), and the selective drugs from the non-selective drugs. Our work suggests a new way of multidimensional drug screening and target identification, which has significant potential applications for drug discovery and design.

Optimization of cyclotide extraction parameters

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds, including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50% MeOH for 6h at a plant material to solvent ratio of 0.5:10 (g/mL) represents an optimum extraction method.

Fluorescence-profile pre-definable quantum-dot barcodes in liquid-core microcapsules

Microparticles incorporated with quantum-dot (QD) barcodes for multiplexed bioassays attract a great attention due to their potential applications in drug discovery, gene profiling and clinic diagnostics. However, the existing QD barcodes lack a necessary optical stability to ambient fluids or a repeatability of fluorescent profiles. We developed a new QD barcode by loading an aqueous QD mixture as a liquid core into a monodispersed polymer microcapsule by a microfluidic method to avoid those problems. We found that the QDs in the liquid cores were able to maintain their original characteristics, especially the linear relation between photoluminescence intensity and concentration. In addition, we found that the fluorescent profiles of the QD-loaded liquid cores were the same as those of the QD mixtures before being loaded inside the microcapsules. With these two properties, the QD barcodes can be predefined directly by multiplexing the emission peaks and concentrations of the QDs in the liquid cores. Furthermore, the graphical information from fluorescent images of the microcapsules, such as the sizes and numbers of the QD-loaded liquid cores, offers another dimension for barcoding to increase the coding capacity. We also presented a microfluidic method to manufacture the QD-barcoded microcapsules of size ~30 μm. These pre-definable QD barcodes with stable fluorescent profiles can be used as a platform for various high-throughput screening applications in different bioassay buffers.

Chemical proteomics: terra incognita for novel drug target profiling

The growing demand for new therapeutic strategies in the medical and pharmaceutic fields has resulted in a pressing need for novel druggable targets. Paradoxically, however, the targets of certain drugs that are already widely used in clinical practice have largely not been annotated. Because the pharmacologic effects of a drug can only be appreciated when its interactions with cellular components are clearly delineated, an integrated deconvolution of drug-target interactions for each drug is necessary. The emerging field of chemical proteomics represents a powerful mass spectrometry (MS)-based affinity chromatography approach for identifying proteome-wide small molecule-protein interactions and mapping these interactions to signaling and metabolic pathways. This technique could comprehensively characterize drug targets, profile the toxicity of known drugs, and identify possible off-target activities. With the use of this technique, candidate drug molecules could be optimized, and predictable side effects might consequently be avoided. Herein, we provide a holistic overview of the major chemical proteomic approaches and highlight recent advances in this area as well as its potential applications in drug discovery.

Effects of Substrate Mechanics on Contractility of Cardiomyocytes Generated from Human Pluripotent Stem Cells

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4–99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.

Lensfree on-chip holography facilitates novel microscopy applications

Lensfree on-chip holography facilitates novel microscopy applications

Automated Lipid Bilayer Formation using a PDMA Gasket

Planar lipid bilayer membranes(BLMs) play important roles in studying ion channels, as well as in potential applications for drug discovery. Despite the importance of many practical applications using BLMs, membrane formation is still based on conventional methods invented by Montal and Mueller in 1960s. Although membranes can be simply reconstituted using the conventional technique, membranes should be created where experiments are conducted due to their mechanical instability. In our recent work a membrane formation technique using high melting temperature solvent mixture was devised by Jeon, et al.

Revealing promiscuous drug–target interactions by chemical proteomics

The (poly-)pharmacological activities of a drug can only be understood if its interactions with cellular components are comprehensively characterized. Mass spectrometry-based chemical proteomics approaches have recently emerged as powerful tools for the characterization of drug-target interactions in samples from cell lines and tissues. At the same time, off-target activities can be identified. This information can contribute toward optimization of candidate drug molecules and reduction of side effects. In this review, we describe recent advances in chemical proteomics and outline potential applications in drug discovery.

Protein recognition via oligonucleotide-linked small molecules: Utilisation of the hemin-binding aptamer

The development of oligonucleotide-linked small molecules for protein recognition is a worthy undertaking with potential applications in drug discovery and diagnostics. Although the ground work for this novel approach to protein recognition is still being laid, a number of promising systems have recently been developed. Herein, we discuss a selection of these systems focusing on the distinct tactics that can be employed for protein binding. Also reported is a new example that underscores the unique versatility of linking aptamers to protein-binding small molecules. In particular, we discuss the development of a chimeric aptamer that in the ‘on’ state can bind and signal the presence of a target protein. Furthermore, we demonstrate that the activity of this sensing supramolecule can be switched ‘off’ on cue.

Chemical synthesis in nanosized cavities

This account briefly discusses methods of in-cavity synthesis with the main focus on molecularly imprinted polymers and self-assembled capsules. Discussed are examples that highlight recent progress and outline key challenges for the further development of in-cavity synthesis. Emphasis is intentionally placed at potential applications in drug discovery and combinatorial technologies.