Unlocking the potential of chemical probes for methyl-lysine reader proteins.

Abstract

Future Medicinal ChemistryVol. 7, No. 14 Themed Issue: Perspectives from Academic Drug Discovery Conference (Cambridge, UK) – EditorialsFree AccessUnlocking the potential of chemical probes for methyl-lysine reader proteinsStephen V FryeStephen V Frye*Author for correspondence: E-mail Address: svfrye@email.unc.edu Division of Chemical Biology & Medicinal Chemistry, Center for Integrative Chemical Biology & Drug Discovery, Eshelman School of Pharmacy, 125 Mason Farm Road, 3012 Marsico Hall, UNC-Chapel Hill, NC 27599-7363, USASearch for more papers by this authorPublished Online:22 Sep 2015https://doi.org/10.4155/fmc.15.119AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Interest in the role of chromatin regulatory proteins in human biology and disease has grown enormously over the last 15 years. Chromatin is the complex of histone proteins, RNA and DNA that efficiently packages the genome within each eukaryotic cell. While cell lineage specific transcription factors clearly play a dominant role in the control of gene expression [1], the regulation of chromatin accessibility via post-translational modifications (PTM) of histones is of great current interest as the opportunities for pharmacological intervention are significantly better than direct perturbation of transcription factors [2]. Our understanding of chromatin regulation is in its infancy and chemical biology is poised to play a central role in advancing scientific knowledge and assessing therapeutic opportunities in this field. Specifically, cell penetrant, high-quality chemical probes that influence chromatin state are of great significance [3]. The advantages of a small-molecule driven approach to modulating chromatin biology are numerous: temporal resolution; mechanistic flexibility (targeting a specific activity of a protein as opposed to ablating them all in transgenic knock-outs or DNA editing and RNA-interference techniques); ease of delivery; and most significantly, a small molecule tool has the potential to provide an immediate transition to a drug discovery effort, possibly cutting years off the time between target validation and therapeutic intervention [4].While the enzymes that perform PTMs on histones are an important and precedented class of druggable targets [2,5], the biological consequences of many PTMs result from their recruitment of regulatory machinery via protein–protein interactions directly facilitated by the PTM. The binding domains involved in PTM recognition on chromatin are referred to as ‘readers.’ We have been focused on exploration of the druggability of readers of methyl-lysine (Kme) as this PTM plays a central role in chromatin regulation and more than 200 Kme reader domains within several protein families occur within the human proteome – making this a large and relatively unexplored target-class for probe discovery [6–11].The primary intent of a chemical probe is to establish the relationship between a molecular target, usually a protein whose function is modulated by the probe, and the biological consequences of that modulation. In order to fulfill this purpose, a chemical probe must be profiled for selectivity, mechanism of action and cellular activity, as the cell is the minimal system in which ‘biology’ can be explored [3,4,12]. Fortuitously, the scientific community's interest in chromatin regulation and the appreciation for the role of chemical probes in driving biological understanding has intersected in an exciting and productive fashion recently resulting in rapid translation of probe-enabled biological hypotheses to clinical interventions [2]. While inhibitors of druggable enzyme targets such as the protein lysine methyltransferases, EZH2 and DOT1L have followed on the heels of histone deacetylases inhibitors in pioneering clinical studies of small molecules that regulate histone PTMs [5], small molecule antagonists of the readers of acetyl-lysine (Kac, bromodomains) are perhaps the poster child for the ‘probe-to-biology-to-drug’ revolution in the field of chromatin regulation. In a period of less than 5 years, chemical probes for bromodomains were described [13,14], their ability to regulate the c-Myc oncogene defined [15,16], and large pharmaceutical companies invested significantly in drug development in this area (e.g., Merck purchased OncoEthix for US$375 million for a Phase 1b compound, OTX-015, that is an amide derivative of the original chemical probe [13] published by the Structural Genomics Consortium [SGC]) [17]. The critical insight that the ‘undruggable’ transcription factor and oncogene, c-Myc, could be modulated in a therapeutically useful fashion was driven by the activity of the initial chemical probes, JQ1 and I-BET [13,14]. In addition to this translational impact, investigation of the mechanism of c-Myc modulation led to a fundamentally new concept in chromatin regulation – the existence of super-enhancer sites that are uniquely responsive to disruption of BRD4 bromodomain recognition of Kac [18]. Readers of Kme are poised to be the next significant target-class integral to chromatin regulation to yield validated and derisked drug targets based on chemical probe exploration.Chemical strategies underlying probe design for Kme readers are still developing, and will not be discussed here. Instead I will discuss validation of chemical probes for this new target class. Characterization of selectivity and cellular target engagement are both essential aspects of probe validation and can be challenging for proteins that mediate protein–protein interactions. In the case of the enzymes that regulate chromatin state, a knockdown of the target by siRNA or shRNA directly perturbs a PTM that can be readily monitored at either a global level or at a specific gene locus [2,5]. Comparison of the effect of such a knockdown to the effect of an inhibitor of the same enzyme then leads to a rather direct biochemical assessment of whether the small molecule is potently and selectively engaging its target in the cell. For Kme readers, siRNA or shRNA knockdowns or genetic manipulations in whole organisms tend to result in phenotypic outcomes that are less easily attributed to specific biochemical changes at the level of chromatin. Additionally, since most Kme readers occur in the context of multidomain and hence multifunctional proteins, there is no a priori basis to expect that pharmacologic antagonism of the Kme reader function will be equivalent to the removal of the whole protein in which it is embedded. For this reason, initial assessments of chromatin reader antagonism have frequently relied upon the effect of the small-molecule ligand on the localization or mobility of a tagged version of its reader target expressed in a cell of interest. This approach has been applied to bromodomains [13,14] and Kme readers in our own work [8]. Fluorescence recovery after photobleaching (FRAP) is a standard technique to detect the effect of small molecule reader antagonists on the rate at which unbleached copies of the tagged Kme reader diffuse into a bleached region of the cell nucleus. While this assay gives a readout that is both proximal to chromatin, and logically attributable to the likely mechanism of action of the ligand (much like changes in PTM levels due to enzyme inhibitors), this phenomenology is difficult to relate to any specific biological function of the endogenous reader and does not directly establish a molecular pathway connection to phenotypic effects [11]. New technologies to assess cellular target engagement are of great interest in chemical biology and could have a significant impact on validation of Kme reader antagonists [19].Selectivity assessment is perhaps the most important aspect of chemical probe characterization, and unfortunately, one that is often lacking in the literature [3,4,12]. While single-target specificity is not an absolute requirement, sufficient profiling data to confidently attribute in vivo effects to the in vitro profile of a probe are essential. We have attempted to address this (in collaboration with the Bedford laboratory, MD Anderson) for Kme readers by determining the binding of biotinylated versions of Kme reader probes to a nitro-cellulose membrane upon which hundreds of potential Kme reader domains have been spotted. Binding is then observed with a streptavidin–dye conjugate and positive results followed up in solution by isothermal titration calorimetry [8]. A potential weakness of this approach is that while we can validate that the position of biotin tagging does not significantly reduce on-target potency, we cannot be assured that the tag does not lead to false negatives in the binding assay. A label-free method of economically assessing chemical probe profiles versus a large panel of Kme readers would represent a significant advance and we continue to focus on this problem. In addition to assessing selectivity versus Kme reader proteins, probes must be profiled versus the enzyme families that modify lysine (protein lysine methyltransferases, lysine demethylases), as activity here would be likely to confound interpretation of both chromatin biochemical readouts and phenotypic outcomes. Profiling versus general pharmacology panels (GPCRs, ion channels, protein kinases) is also performed in order to create a more complete assessment of potential off-target activities. Therefore, our Kme chemical probes are tested versus hundreds of chromatin-related proteins as well as hundreds of diverse pharmacological targets. While these data cannot rule out contributions from unexamined or unknown protein off-targets to a probe's activity, it does support the case for specificity when cellular target-engagement has additionally been proven and would, perhaps, satisfy Bertrand Russell: “If one holds that nothing is certain one must, I think, also admit that some things are much more nearly certain than others.”The pursuit of well validated chemical probes for Kme reader proteins represents a novel, emerging area that will create new understanding of chromatin biology and prove useful in validation of Kme readers implicated in disease. As this target-class is well represented in databases of proteins genetically altered in cancer and neurological disease, the opportunity for translational impact is promising. Additionally, we are committed to developing new approaches for Kme reader probe characterization and freely sharing high-quality probes. Momentum is building in the chemical biology community to create a central source for ‘best-in-class’ probes and we hope that Kme reader probes will one day be well represented [20].AcknowledgementsThe author wishes to recognize the collaborative contributions of all co-authors in our cited references and especially the Structural Genomics Consortium.Financial & competing interests disclosureThe research described here was supported by the National Institute of General Medical Sciences, US National Institutes of Health (NIH, grant RC1GM090732 and R01GM100919), the Carolina Partnership and the University Cancer Research Fund, and University of North Carolina at Chapel Hill. 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Biol. 11(8), 536–541 (2015).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByEngineering a methyllysine reader with photoactive amino acid in mammalian cells1 January 2020 | Chemical Communications, Vol. 56, No. 81Site-specific azide-acetyllysine photochemistry on epigenetic readers for interactome profiling1 January 2017 | Chemical Science, Vol. 8, No. 6Chemical probes for methyl lysine reader domainsCurrent Opinion in Chemical Biology, Vol. 33Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform17 February 2016 | Nucleic Acids Research, Vol. 44, No. 9 Vol. 7, No. 14 Follow us on social media for the latest updates Metrics History Published online 22 September 2015 Published in print September 2015 Information© Future Science LtdAcknowledgementsThe author wishes to recognize the collaborative contributions of all co-authors in our cited references and especially the Structural Genomics Consortium.Financial & competing interests disclosureThe research described here was supported by the National Institute of General Medical Sciences, US National Institutes of Health (NIH, grant RC1GM090732 and R01GM100919), the Carolina Partnership and the University Cancer Research Fund, and University of North Carolina at Chapel Hill. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download