scholarly journals Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR

Molecules ◽  
2019 ◽  
Vol 24 (18) ◽  
pp. 3352 ◽  
Author(s):  
Carlos Alvarado ◽  
Erik Stahl ◽  
Karissa Koessel ◽  
Andrew Rivera ◽  
Brian R. Cherry ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Focal Adhesion ◽  
High Throughput Screening ◽  
Quantum Coherence ◽  
Focal Adhesions ◽  
Scaffolding Protein ◽  
Screening Assay ◽  
Binding Interface ◽  
Novel Approach ◽  
Fragment Library

The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays—a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.

scholarly journals Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction

2019 ◽  
Vol 25 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Timothy Marlowe ◽  
Carlos Alvarado ◽  
Andrew Rivera ◽  
Felicia Lenzo ◽  
Rohini Nott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Focal Adhesion ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Drug Target ◽  
Scaffolding Protein ◽  
Cancer Drug ◽  
Screening Assay ◽  
High Throughput Screening Assay

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

Development of a high-throughput screening assay for drug discovery in SOD1-mediated ALS

2009 ◽  
Vol 65 ◽  
pp. S120
Author(s):  
Gaku Murakami ◽  
Haruhisa Inoue ◽  
Kayoko Tsukita ◽  
Yasuyuki Asai ◽  
Kazuhiro Aiba ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin

1999 ◽  
Vol 112 (2) ◽  
pp. 181-190 ◽  
Author(s):  
S.M. Thomas ◽  
M. Hagel ◽  
C.E. Turner
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Negative Regulator ◽  
Scaffolding Protein ◽  
Minor Role ◽  
Lim Domains ◽  
Focal Adhesion Protein ◽  
A Minor

Paxillin is a focal adhesion scaffolding protein which was originally identified as a substrate of the oncogenic tyrosine kinase, v-src. Paxillin has been proposed to be involved in regulation of focal adhesion dynamics. Two alternatively spliced mouse paxillin cDNAs were cloned and in the process, a paxillin-related protein, Hic-5, was also identified. Cloning and characterization of Hic-5 indicates that this protein shares extensive homology with paxillin. Although Hic-5 was originally characterized as a TGF-beta-inducible gene and proposed to be a transcription factor involved in senescence, the studies here demonstrate that Hic-5 is localized to focal adhesion in REF52 cells and can interact with the focal adhesion proteins, Fak, Frnk, and vinculin. In addition, like paxillin, Hic-5 can bind to a negative regulator of Src PTKs, csk but does not bind to the adaptor protein Crk. Like paxillin, localization of this protein to focal adhesions is mediated primarily by the LIM domains; however, sequences outside the LIM domains also play a minor role in focal adhesion targeting. These results suggest that Hic-5 like paxillin could be involved in regulation of focal adhesion dynamics and raise the possibility that Hic-5 and paxillin could have overlapping or opposing functions in the overall regulation of cell growth and differentiation.

scholarly journals Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates

2003 ◽  
Vol 8 (4) ◽  
pp. 453-462 ◽  
Author(s):  
Li Di ◽  
Edward H. Kerns ◽  
Yan Hong ◽  
Teresa A. Kleintop ◽  
Oliver J. Mc Connell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Parent Compound ◽  
Metabolic Stability ◽  
Screening Assay ◽  
Drug Candidates ◽  
Increasing Demand ◽  
The Stability

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.

A high-throughput screening assay for drug discovery in SOD1-mediated ALS targeting the transcription of SOD1

2010 ◽  
Vol 68 ◽  
pp. e311
Author(s):  
Gaku Murakami ◽  
Haruhisa Inoue ◽  
Kayoko Tsukita ◽  
Yasuyuki Asai ◽  
Kazuhiro Aiba ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Predicting Targeted Cancer Therapeutics

2016 ◽  
Author(s):  
Aaron Wise ◽  
Murat Can Cobanoglu
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Cancer Therapeutics ◽  
Predictive Performance ◽  
Training Data ◽  
Brute Force ◽  
Functional Assays ◽  
Novel Approach

AbstractMotivation: Cancer is a complex and evolving disease, making it difficult to discover effective treatments. Traditional drug discovery relies on high-throughput screening on reductionist models in order to enable the testing of 105 or 106 compounds. These assays lack the complexity of the human disease. Functional assays overcome this limitation by testing drugs on human tumors, however they can only test few drugs, and remain restricted to diagnostic use. An algorithm that identifies hits with fewer experiments could enable the use of functional assays for de novo drug discovery.Results: We developed a novel approach that we termed ‘algorithmic ideation’ (AI) to select experiments, and demonstrated that this approach discovers hits 104 times more effectively than brute-force screening. The algorithm trains on known drug-target-disease associations assembled as a tensor, built from the (public) TCGA and STITCH databases and predicts novel associations. We evaluated our tensor completion approach using a temporal cutoff with data prior to 2012 used as training data, and data from 2012 to 2015 used as testing data. Our approach achieved 104-fold more efficient hit discovery than the traditional brute-force high-throughput screening. We further tested the method in a sparse, low data regime by removing up to 90% of the training data, and demonstrated the robustness of the approach. Finally we test predictive performance on drugs with no previously known interactions, and the algorithm demonstrates 103-fold improvement in this challenging problem. Thus algorithmic ideation can potentially enable targeted antineoplastic discovery on functional assays.Availability: Freely accessible at https://bitbucket.org/aiinc/drugx.Contact:[email protected], [email protected]

scholarly journals Activation of Rho-Dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

1998 ◽  
Vol 18 (5) ◽  
pp. 3044-3058 ◽  
Author(s):  
Zeynep F. Altun-Gultekin ◽  
Sanjay Chandriani ◽  
Cecile Bougeret ◽  
Toshimasa Ishizaki ◽  
Shuh Narumiya ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Focal Adhesion ◽  
Lysophosphatidic Acid ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Stress Fibers ◽  
Scaffolding Protein ◽  
Actin Cables

ABSTRACT The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, α3-integrin, and a higher-molecular-weight form of β1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

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