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Scientific Articles

Saxena C., Identification of protein binding partners of small molecules using label-free methods. , Expert Opinion on Drug Discovery (2016)11:1017.1025.

Different Label-free Chemical Proteomics Technologies for Target Identification are making in-roads. This review describes some of the major technologies and argue advantages of Shantani's label-free Unique Polymer Technologies (UPT) over other methods.

Hati S., Spiro[pyrrolidine-3, 3´-oxindole] as potent anti-breast cancer compounds: Their design, synthesis, biological evaluation and cellular target identification , Nature-Scientific Report(2016)6:32213.

In this work Unique Polymer Technology (UPT) was utilized to identify the targets of newly synthesized anti-cancer compound Spiro[pyrrolidine-3, 3´-oxindole]. Publcations outlines method and workflow of UPT.

Bathula C., Synthesis of novel 5-arylidenethiazolidinones with apoptotic properties via a three component reaction using piperidine as a bifunctional reagent , Org Biomol Chem (2016)14:8053-63.

In this work Unique Polymer Technology (UPT) was utilized to identify the targets of newly synthesized derivative of thiazolidinones. UPT identified beta actin like protein 2 (Uniprot ID Q562R1), gamma-enolase (Uniprot ID P09104) and macrophage migration inhibitory factor (Uniprot ID P14174) as putative targets of the compound.

Saxena C., Affinity-Based Chemoproteomics with Small Molecule-Peptide Conjugates , Methods Mol Biol (2012) 803:39-54.

In affinity-based chemoproteomics strategies, the direct immobilization of small bioactive probe molecules to a solid support may pose problems with respect to the preservation of the functional activity toward the target proteins. Typically, immobilized molecules on solid supports exhibit lower affinity for target proteins compared to the free parent molecule. This may lead to a failure to specifically capture the target proteins or to unacceptable losses during the washing steps. To circumvent these shortcomings, we have devised small molecule-peptide conjugates (SMPCs), which enable wide-ranging experimental strategies for the capturing of protein targets of small molecules from cells or tissues. With the possibilities of synthesizing peptides of tailored biochemical and biophysical properties, SMPCs enable the identification of protein targets of small molecules from cell-lysates and intact cells. Moreover, labeling of these conjugates with fluorophores can provide information on the cellular localization and distribution of the target.

Saxena et al, Capture of Drug Targets from Live Cells Using a Multipurpose Immuno-Chemo-Proteomics Tool , J. Proteome Res. (2009) 8:3951-3957.

Recently we have described the development of an Immuno-chemo-proteomics method for drug target deconvolution and profiling the toxicity of known drugs (Saxena, C.; Zhen, E.; Higgs, R. E.; Hale, J. E.J. Proteome Res.2008, 8, 3490−3497). The orthogonal nature and advantage of the newly developed method over existing ones were presented. Most commonly, a small molecule was coupled to an epitope and used as an affinity probe to bind targets and later antibody against the epitope was used to isolate the probe-protein complex. However, such studies performed using cell lysates are prone to false positive identification because the protein source is not in its native physiological condition. Here we describe the development and application of a multipurpose soluble probe where a small molecule was coupled to a fluorophore-tagged cell-permeable peptide epitope, which was used to affinity isolate binding proteins from live cells. Fluorophore coupling allowed direct visualization of the compound in the cells, and cell permeability of the probe provided opportunity to capture the targets from the live cell. The GSK3-β inhibitor Bisindolylmaleimide-III was coupled to a peptide containing the fluorescein-tagged TAT epitope. Following incubation with the live cells, the compound and associated proteins were affinity isolated using antifluorescein antibody beads. Using this approach, we captured the known Bisindolylmaleimide-III target GSK3-β and previously unidentified targets from live cells. Dose-dependent inhibition of target binding to probe in the presence of uncoupled compound validated the approach. This method was directly compared with the one where cell lysate was used as the protein source providing an advanced strategy to aid in target deconvolution and help to eliminate false positives originating from non-native protein source.

Saxena C., Small-molecule affinity chromatography coupled mass spectrometry for drug target deconvolution , Expert Opinion on Drug Discovery (2009) 4:701- 714.

Background: Current drug discovery organizations have renewed interest in phenotypic/function based screening for the identification of novel small-molecule drug candidates. Phenotypic screening faces the challenge of deconvoluting the identity of molecular targets of small-molecules through which they exert their biological effect. The identity of the target is crucial for understanding the mechanism of drug action, rational drug design, interpretation of any toxicological findings and patient stratification. Several methods are available to deconvolute the targets of small-molecules. Objective: This review describes successful examples, limitations and advances of drug target deconvolution using small-molecule affinity chromatography coupled mass spectrometry based methods. A brief discussion of other target deconvolution methods is also presented for comparative appreciation of mass spectrometry based methods. Conclusion: The use of small-molecule affinity chromatography coupled mass spectrometry based methods is gaining popularity as a technique for target identification. Mass spectrometry based methods provide fast, reliable and high-content information on the target. They can be used with relatively intact biological systems to develop a system-wide understanding of the drug-target interaction.

Saxena et al, An Immuno-Chemo-Proteomics Method for Drug Target Deconvolution , J. Proteome Res. (2008) 7:3490-3497.

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-α inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-α, GSK3-β, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-α, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.