Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of in-vitro activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target's role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase).

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1-313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.

Drug design for protein kinases and phosphatases: flexible-receptor docking, binding affinity and specificity, and drug-binding kinetics.

This article reviews some of our experiences on applying computational techniques to aid the design of drugs targeting protein kinases and phosphatases. It is not a comprehensive review. Rather, it focuses on several less explored approaches or ideas that we have experiences on. It reviews some recent improvements on the Poisson-Boltzmann/Surface Area model for calculating binding affinity and discusses ways to perform calculations that are more tolerant to statistical and systematic errors. Several new ways to incorporate protein flexibility in molecular docking and estimating binding affinity are also discussed. Its discussions also go beyond binding affinity to considering drug-binding kinetics, not only on investigating protein-ligand interactions in isolation, but also on accounting for upstream and downstream influences that can occur in cells, through kinetic modeling of cell signaling. This review also describes a quick molecular simulation method for understanding drug-binding kinetics at the molecular level, with the hope of generating guiding principles for designing drugs with the desired kinetic properties. Sources of drug-binding selectivity that appear obvious but often overlooked are also discussed.

Influence of kinetics of drug binding on EGFR signaling: a comparative study of three EGFR signaling pathway models.

We used three models of the epidermal growth factor receptor (EGFR) signaling pathway mimicking three different cell lines to study the effects of kinetics of drug binding on influencing molecular signaling in the pathways. With no incubation of drugs before the external cue epidermal growth factor (EGF) was applied, we found that fast kinetics of binding to protein kinases was advantageous in suppressing the production of the Extracellular signal-regulated kinase (ERK) that triggers cell proliferation, with some exceptions. Incubation of a drug with a protein kinase target for an hour before a pathway was initiated with an external cue made kinetics less significant, so did high concentration of drugs. In addition, we found that applying a drug to a protein kinase mostly affected downstream signaling although upstream events were also affected in a few cases. In examining whether applying two drugs to two protein kinase targets in the pathways could produce synergistic effects, we found positive, negative, or no effects, depending on the protein kinases targeted and the pathway model considered.