{"id":544,"date":"2020-01-06T08:24:38","date_gmt":"2020-01-06T08:24:38","guid":{"rendered":"https:\/\/zobio.com\/online\/?p=544"},"modified":"2023-03-09T10:02:03","modified_gmt":"2023-03-09T10:02:03","slug":"ythdf2-an-unprecedented-epitranscriptomic-reader-protein","status":"publish","type":"post","link":"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/","title":{"rendered":"YTHDF2 \u2013 An unprecedented epitranscriptomic reader protein"},"content":{"rendered":"<div id=\"pl-544\"  class=\"panel-layout\" ><div id=\"pg-544-0\"  class=\"panel-grid panel-has-style\" ><div class=\"panel-row-style panel-row-style-for-544-0\" ><div id=\"pgc-544-0-0\"  class=\"panel-grid-cell\" ><div id=\"panel-544-0-0-0\" class=\"so-panel widget widget_sow-editor panel-first-child panel-last-child\" data-index=\"0\" ><div class=\"with-icon panel-widget-style panel-widget-style-for-544-0-0-0\" ><div\n\t\t\t\n\t\t\tclass=\"so-widget-sow-editor so-widget-sow-editor-base\"\n\t\t\t\n\t\t><h2 class=\"widget-title\">YTHDF2 \u2013 A unprecedented epitranscriptomic reader protein<\/h2>\n<div class=\"siteorigin-widget-tinymce textwidget\">\n\t<p><b>Challenge<\/b><br \/>\nThe epitranscriptomic YTHDF2 reader is a member of a family of proteins that selectively binds to a modified base (m6A) in mRNA.\u00a0 Our client tasked us with setting up a complete \u201cgene to lead\u201d fragment-based drug discovery campaign. Since the entire family is unprecedented, there were no \u201ctool compounds\u201d typically required to set up assays. As we progressed, further challenges presented themselves.<\/p>\n<p><strong><span lang=\"EN-US\">Solution<br \/>\n<\/span><\/strong>Once we determined that we could immobilize YTHDF2 while retaining its selective m6A binding, we could use our proprietary TINS fragment screening technology, which does not require tool compounds to setup. TINS generated a list of primary hits from the ZoBio fragment library. Using a nearly identical immobilization approach, we observed binding of many of these raw hits in SPR. The binding response could be used to optimize the assay for fragment screening.<\/p>\n<p>We subsequently confirmed that hits from both TINS and SPR targeted the desired binding site on YTHDF2 using protein observed NMR experiments. This information rich assay revealed that buffer components were also selectively binding to the desired site and effectively competing against the fragment hits. With this knowledge we could further optimize our assays and structural biology approaches.<\/p>\n<p><strong>Inverse SPR assay<\/strong><br \/>\nThe classic \u201cforward\u201d SPR assay identified compounds that selectively bind to YTHDF2 with ideal behavior. However, this assay does not provide insight into the biological activity of compounds, that is whether or not they prevent binding of m6A containing RNA. In order to do so, we developed an inverse SPR assay in which RNA carrying the relevant chemical modification, is immobilized. This allows us to directly probe the ability of test compounds to interfere with the binding of YTHDF2 to the immobilized RNA, in other words, a biophysics-based biological assay. We further refined the assay to quantify the level of competition based on the affinity of the ligand to discriminate and prioritize chemotypes.<\/p>\n<p><strong>The assay cascade<\/strong><br \/>\nWe efficiently guided medicinal chemistry efforts by focusing on functionally active chemotypes, as determined by iSPR. These compounds were further characterized by biochemical assays (trFRET). Chemotypes exhibiting good correlation in the different assays were prioritized. From the start of the project, NMR played a crucial role in obtaining structural information about the way selected chemotypes bound the target. Initially we could rapidly map the binding sites of 10\u2019s of compounds at low resolution. Subsequently, when it was not possible to obtain liganded structures do to crystal packing issues, we elucidated the solution structure of ligand-protein complexes that were then used to guide medicinal chemistry.<\/p>\n<p><b>Achievements<\/b><br \/>\nBy capitalizing on our unique expertise in developing customized biophysical assays, we created a biologically relevant compound screening cascade in the absence of tool compounds. The cascade delivered chemically diverse sets of validated fragments that we successfully elaborated towards lead compounds using structural information that could only be gleaned through NMR.<\/p>\n<\/div>\n<\/div><\/div><\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"<p><b>Challenge<\/b><br \/>\nOur client tasked us with setting up a complete \u201cgene to lead\u201d fragment-based drug discovery campaign.<br \/>\nSince the entire family is unprecedented, there were no \u201ctool compounds\u201d typically required to set up assays. As we progressed, further challenges presented themselves. <\/p>\n<p><b>Achievements<\/b><br \/>\nBy capitalizing on our unique expertise in developing customized biophysical assays, we created a biologically relevant compound screening cascade in the absence of tool compounds. The cascade delivered chemically diverse sets of validated fragments that we successfully elaborated towards lead compounds using structural information that could only be gleaned through NMR.<\/p>\n","protected":false},"author":3,"featured_media":3989,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"gpsa_enabled":false,"gpsa_required_form_ids":[],"gpsa_require_unique_form_submission":false,"gpsa_access_behavior":"show_message","gpsa_form_redirect_path":"","gpsa_requires_access_message":"","gpsa_access":[],"gpsa_content_loading_message":"","footnotes":""},"categories":[5],"tags":[],"class_list":["post-544","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-cases"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.1.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>YTHDF2 \u2013 An unprecedented epitranscriptomic reader protein - ZoBio - Drug Discovery Technology<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"YTHDF2 \u2013 An unprecedented epitranscriptomic reader protein - ZoBio - Drug Discovery Technology\" \/>\n<meta property=\"og:description\" content=\"Challenge Our client tasked us with setting up a complete \u201cgene to lead\u201d fragment-based drug discovery campaign.  Since the entire family is unprecedented, there were no \u201ctool compounds\u201d typically required to set up assays. As we progressed, further challenges presented themselves.   Achievements By capitalizing on our unique expertise in developing customized biophysical assays, we created a biologically relevant compound screening cascade in the absence of tool compounds. The cascade delivered chemically diverse sets of validated fragments that we successfully elaborated towards lead compounds using structural information that could only be gleaned through NMR.\" \/>\n<meta property=\"og:url\" content=\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\" \/>\n<meta property=\"og:site_name\" content=\"ZoBio - Drug Discovery Technology\" \/>\n<meta property=\"article:published_time\" content=\"2020-01-06T08:24:38+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2023-03-09T10:02:03+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/zobio.com\/online\/wp-content\/uploads\/2020\/01\/JV7A9921-1-e1678356118107.jpg\" \/>\n\t<meta property=\"og:image:width\" content=\"2000\" \/>\n\t<meta property=\"og:image:height\" content=\"1100\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/jpeg\" \/>\n<meta name=\"author\" content=\"Content manager ~KB\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"Content manager ~KB\" \/>\n\t<meta name=\"twitter:label2\" content=\"Estimated reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/#article\",\"isPartOf\":{\"@id\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\"},\"author\":{\"name\":\"Content manager ~KB\",\"@id\":\"https:\/\/zobio.com\/online\/#\/schema\/person\/fcdd542fb16960e243f01d731e59d707\"},\"headline\":\"YTHDF2 \u2013 An unprecedented epitranscriptomic reader protein\",\"datePublished\":\"2020-01-06T08:24:38+00:00\",\"dateModified\":\"2023-03-09T10:02:03+00:00\",\"mainEntityOfPage\":{\"@id\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\"},\"wordCount\":485,\"publisher\":{\"@id\":\"https:\/\/zobio.com\/online\/#organization\"},\"image\":{\"@id\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/#primaryimage\"},\"thumbnailUrl\":\"https:\/\/zobio.com\/online\/wp-content\/uploads\/2020\/01\/JV7A9921-1-e1678356118107.jpg\",\"articleSection\":[\"Cases\"],\"inLanguage\":\"en-GB\"},{\"@type\":\"WebPage\",\"@id\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\",\"url\":\"https:\/\/zobio.com\/online\/cases\/ythdf2-an-unprecedented-epitranscriptomic-reader-protein\/\",\"name\":\"YTHDF2 \u2013 An unprecedented epitranscriptomic reader protein - 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