Citation: Martin KR, Narang P, Xu Y, Kauffman AL, Petit J, et al. (2012) Identification of Small Molecule Inhibitors of PTPs through an Integrative Virtual and Biochemical Approach. Editor: Andreas Hofmann, Griffith University, Australia Received January 12, 2012; Accepted October 22, 2012; Published November 20, 2012 Copyright: ?2012 Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Department of Defense Prostate Cancer Research Program of the Office of Congressionally Directed Medical Research Programs PC081089 to JPM. JPM is also supported by Award Number R01CA138651 from the National Cancer Institute. Award Number RC2MH090878 from the National Institute of Mental Health awarded to NM provided partial support for the work described in this paper. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Tyrosine phosphorylation is a critical mechanism by which cells exert control over signaling processes. Protein tyrosine kinases (PTKs) and phosphatases (PTPs) work in concert to control these signaling cascades, and alterations in the expression or activity of these enzymes hallmark many human diseases [1,2]. While PTKs have long been the focus of extensive research and drug development efforts, the role of PTPs as critical mediators of signal transduction was initially underappreciated [3]. Consequently, the molecular characterization of these phosphatases has trailed that of PTKs, and only recently has the PTP field reached the forefront of disease based-research. As validation for phosphastases in human disease, half of PTP genes are now implicated in at least one human disease [3]. The critical role of PTPs in cell function and their role in disease etiology highlight the importance of developing phosphatase agonists and inhibitors. Unfortunately, phosphatases have historically been perceived as “undruggable” for several important reasons [4]. First, phosphatases often control multiple signaling pathways and thus, inhibition of a single enzyme may not yield a specific cellular effect. Second, signaling cascades are generally controlled by multiple phosphatases and accordingly, blocking the activity of one may not sufficiently induce the desired modulation to a signaling pathway. Finally, and most importantly, phosphatase active sites display high conservation which hinders the ability to develop catalysis-directed inhibitors with any degree of selectivity [4]. Despite these pitfalls, the emerging role of PTPs in human disease etiology has necessitated a solution. Largely through use of structure-based drug design, several PTPs now represent promising targets for disease treatment. Most notably, bidentate inhibitors of PTP1B, implicated in type II diabetes and obesity, have been developed which span both the catalytic pocket and a second substrate binding pocket discovered adjacent to the active site [5,6,7]. Drug development around PTP1B has provided a proof-ofconcept for investigations focused on additional PTP targets. Several studies have uncovered physiologically important and disease relevant functions for the classic receptor type PTP, PTPs (encoded by the PTPRS gene), which underscore its potential as a biological target. PTPs is highly expressed in neuronal tissue where it regulates axon guidance and neurite outgrowth [8,9,10,11,12]. Furthermore, it was recently reported that loss of PTPs facilitates nerve regeneration following spinal cord injury (SCI), owing to the interaction of its ectodomain with chondroitin sulfate proteoglycans (CSPGs) [13,14]. In addition to its neural function, PTPs has been implicated in chemoresistance of cancer cells. First, we discovered that RNAi-mediated knockdown of PTPs in cultured cancer cells confers resistance to several chemotherapeutics [15]. Additionally, we have discovered that loss of PTPs hyperactivates autophagy, a cellular recycling program that may contribute to chemoresistance of cancer cells [16]. Taken together, it is apparent that modulation of PTPs may have therapeutic potential in a range of contexts. Notably, inhibition of PTPs could potentially provide benefit following SCI through enhanced neural regeneration. In addition, it is possible that PTPs inhibition may yield therapeutic value in diseases in which increasing autophagy represents a promising treatment strategy (i.e., neurodegenerative diseases). Furthermore, a small molecule would provide value as a molecular probe or tool compound to interrogate the cellular functions and disease implications of PTPs.
Figure 1. Workflow overview for PTPs inhibitor development. In silico docking was performed using the crystal structure of the D1 active site of PTPs as a target (PDB ID: 2fh7). Three structurally distinct scaffolds were chosen, along with 74 additional compounds identified by a sub-structure search of compounds in the ChemBridge, were tested in vitro to for the ability to inhibit PTPs phosphatase activity. To eliminate oxidation-mediated inhibition, we optimized the biochemical assay and discovered one lead compound whose inhibition was not mediated by oxidation. Future efforts (dashed boxes and lines) will introduce selectivity into this lead compound through a detailed structural analysis of PTPs and related PTPs. Several approaches exist for the identification of small molecule inhibitors of phosphatases. While high-throughput screening (HTS) of compounds in vitro has been successfully utilized to discover inhibitors of LAR (PTPRF), PTP1B, SHP2, CD45, and others [17], the technical and physical investment is considerable as is the potential for experimental artifacts leading to false negatives and positives [17].