Name: drug repurposing hypothesis generation using the "re:fine drugs" system
Uploaded: 2016-12-11T15:00:00.000+00:00
Duration: 5 min 10 s
Description: Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

This work was partially supported by the National Institutes of Health (NIH) Clinical and Translational Science Awards (CTSA) Grant (UL1TR001070) to the Ohio State University's Center for Clinical and Translational Science (CCTS) and the National Library Of Medicine under Award Number T15LM011270.

1.遺伝子からのクエリの開始、薬物または疾患利用規約<ol><li> http://drug-repurposing.nationwidechildrens.org：以下のリンクから：「ファイン薬RE」のホームページにアクセスします。薬（ジェネリック医薬品名）、疾患（新しい病気表示）または遺伝子（公式HGNC遺伝子記号）：以下の3カテゴリーのいずれかからの検索バーに検索タームを入力することによって開始します。 </li><li>唯一の「薬剤名」、「新しい病気の表示」、「遺伝子記号」または「すべて」のカテゴリを含めるように検索バー機能をフィルタリングします。検索バーは、クエリのエントリの自動入力機能を備えています。 </li><li>キーワードを入れて、「検索」ボタンをクリックしてください。次の列のいずれかによって、結果のソート表：「医薬品」、「登録指示」、「P-バリュー」、「調整p値」、「オッズ比」、「研究」、「新表示 &quot;、&quot;医薬品銀行指示」、「Medlineのアブストラクトの＃」、「CLIの＃nicalトライアルレジストリ」、「潜在的な」、「SNP」、「遺伝子」または「アクション」。 </li><li>医薬品情報機能を有効にするために、高度な検索オプションに移動します。特定の薬剤のための「情報」列のアイコンをクリックします。関連のp値、疾患名、薬剤名、遺伝子の詳細（NCBI遺伝子リンク）医薬品の詳細を含むすべての対応する情報（DrugBankリンク）を表示するページを確認します。 </li></ol>詳細オプションの2探査<ol><li>ページの右側にある「高度な」ボタンをクリックし、さらに結果を絞り込むには、いくつかのオプションが提供されています。薬剤、協会、病気、潜在的な、遺伝子およびアクション：高度な検索オプションには、以下の修正が含まれています。 </li><li>エクスポートは、ページの右側にある「エクスポート」ボタンをクリックして、テーブルをもたらします。高度な検索ウィンドウを倒しするために、「シンプル」ボタンをクリックします。&lt;/李&gt; <li>高度なオプション「薬物」タブの下で、特定の薬剤の適応症または結果をフィルタリングするための追加の薬剤名を指定します。 </li><li> 「会合」タブの下で、有意水準p値、FDRで調整p値、効果の大きさ（オッズ比）、および/またはによってフィルタ結果がタイプ（GWAS、PheWASまたは両方）を学びます。 </li><li> 「疾患」タブの下で、予測された新たな使用のための特定の疾患の説明を指定します。 </li><li> 「潜在的」タブで、次の基準のいずれかに記載のフィルタ結果は、（i）薬物指示がDrugBankデータベースに含まれているかどうか、薬剤の共起や病気、とMedlineの抄録の（ii）の数（ ⅲ）薬剤の同時発生と疾患と、（iv）再目的化の可能性を持つClinicalTrials.govデータベースエントリの数を。 注：再目的化可能性のオプションは、発見の新規性について説明します。（ⅰ）既知：関係が既にDrugBankデータベースに存在するが、（ii）は強くサポート：臨床試験レジストリとメド抄録の両方でいくつかのサポート、（iii）の可能性が高い：臨床試験のレジストリでも、Medlineの抄録で証拠：臨床試験のレジストリまたはMedlineの抄録および（iv）小説のいずれかで何らかのサポート。 </li><li> 「遺伝子」タブの下で、特定の薬物標的遺伝子によって結果をフィルタリングするSNP識別子または遺伝子記号を入力します。 </li><li> 「アクション」タブの下で、アゴニスト、アンタゴニスト、他の、未知の、またはすべて（：DrugBankデータベースソース）を含む、薬物標的（複数可）に対する薬物作用のタイプを指定します。 </li></ol>

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The authors declare that they have no competing financial interests.

The protocol described here for the RE:fine Drugs interactive dashboard can be modified in different ways according to the user's preferences. This method uniquely integrates GWAS and PheWAS data as a novel paradigm underlying drug repurposing hypothesis generation. Specifically, this system provides access to both 52,966 PheWAS associations and 7,945 GWAS associations with advanced options to filter the results by the study type, effect size and/or significance level. Another advantage of this method over existing computational drug repurposing tools is that queries may be made from drug, gene or disease perspectives. 
There are several limitations to this method. Currently, the PheWAS data is limited to primarily adult patient population from five institutions contained in the Electronic Medical Records and Genomics (eMERGE) network with a mean age of 69.5 years 12. Additionally, the "repurposing potential" feature uses co-occurrence of search terms in Medline abstracts as one of its criteria. It is well known that text mining methods using co-occurrence have limitations with respect to syntactical structure and literature bias. Thus, we recommend this feature be used as a starting point to explore the potential novelty and/or evidence supporting specific drug repurposing hypotheses and recommend additional investigation into the biomedical literature and clinical trial databases. 
Future directions for this work not described here would be to extend this database to additional sources of GWAS and PheWAS data as they become available. Similar efforts to systematically translate results from large-scale GWAS studies into drug repurposing hypotheses have been previously published 9,13-14. It may be useful to compare these different workflows to predict drug candidates from GWAS data in future studies. Additionally, several other methods exist to computationally generate drug repurposing hypotheses from different data sources, including: genomics, transcriptomics, chemical structures, drug side effect profiles, as previously summarized 6,11. Future methodological advancements could also include automating drug combination predictions and providing information on drug toxicity to guide follow up studies for drug candidates. 
Furthermore, the hypotheses generated from RE:fine Drugs may be further validated using electronic health records, before initiating clinical trials 15. Finally, future studies will be needed to compare this system to other target-based drug repurposing methods.

高スループットの薬物およびリード化合物のスクリーニングを含む伝統的な創薬アプローチに関連したコストがかかり、非効率的なプロセスは、患者1,2のための治療法の研究の発見を翻訳するの遅れに貢献しています。 10億ドルと15年から20年の平均は、ベッドサイド3にベンチから新薬をもたらすために必要とされます。さらに、薬物の52％が第1相臨床試験で開発中に失敗し、フェーズ2に入る化合物の25％のみが完全な第3相臨床試験4に進みます。薬物転用または薬物の再配置の目標は、失敗した麻薬を更新および/またはより高速な患者に新しい治療法を提供し、高い成功率とするために、承認薬のための新規適応症を見つけることです。薬物再利用は3-12年5人の患者に使用するための薬を利用できるようにタイムラインを減少させることができます。薬物再利用のための重要な医療用途は次のとおりです。貧しいprognos伴う疾患をであり、低生存率、薬剤耐性疾患、積立不足病気の研究分野や貧困と不十分な患者集団。 計算薬物転用は、薬剤候補6のための新たな適応症のための仮説を生成することができる自動化されたワークフローを設計および検証するプロセスとして定義されています。既存の計算の薬物再利用方法は、標的ベース、知識ベース、シグネチャベース、ネットワークベースの分類、およびターゲットと機構系、遺伝子、疾患または薬物の視点から配向させることができるされています。また、計算のアプローチは、さらに、概念実証検証実験と再利用薬剤候補7のための小規模な臨床研究を加速することができます。我々は以前、「RE：罰金薬」で報告している、薬物-遺伝子-疾患関係8の推移理論に基づく薬物再利用仮説生成のために自由に利用できる、インタラクティブなWebベースのツール。全体的なグラムこの方法のOALは、体系的、臨床的、業界および規制社会など多様なコミュニティからのユーザーのための薬剤の再利用を可能にするために、薬物の多様な種類、遺伝学的および臨床データを統合することです。このシステムの基本的な方法は、以前にゲノムワイド関連解析（GWAS）と研究9,10を再利用する薬剤でphenomeワイド関連研究（PheWAS）データの使用が報告されています。これらのタイプのデータの新規な組み合わせは、他のターゲットに基づく方法6,11から当社のWebツールを区別します。 RE：罰金薬システムは、現在916薬、567の遺伝子と1770疾患をカバー60911の薬物転用仮説が含まれています。 Webツールは、研究者が対話的に、薬物転用仮説を検索し、多様な基準を使用してそれらに優先順位を付けるためのユーザーフレンドリーなインターフェイスを提供します。たとえば、ユーザーが生物医学文献および臨床試験がdatabaでのサポートを有する薬物転用仮説をフィルタリングすることができますSE、有意なp値、アソシエーションオッズ比または特定の指示によって。このシステムのための唯一の要件は、インターネットへのアクセスです。

<table><tbody><tr><td>Access the homepage for &amp;ldquo;RE:fine Drugs&amp;rdquo; at the following link: http://drug-repurposing.nationwidechildrens.org.&amp;nbsp;</td><td>n/a</td><td>n/a</td><td>The only requirement for this system is Internet access</td></tr></tbody></table>

drug repurposing hypothesis generation using the "re:fine drugs" system

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

この例では、遺伝子「IL2RB」は、遺伝子ベースのクエリとして入力し、自動的にオートフィル機能（ 図1）によってそのように認識されました。 図2に示すように、IL2RB遺伝子について12薬物転用仮説は、返されます。この場合、特定の薬物転用仮説、「ダクリズマブ」の詳細情報ページは、「情報」欄（ 図3）から提供されています。薬物タブが「ダクリズマブ」薬剤に対応するすべての結果によるものであったの図4に示すように結果は、濾過しました。 図5は 、「移植」は、疾患の用語（：DrugBankデータベースソース）のための既知の適応症でのみ薬を示しています。関連]タブには、ユーザーが統計的有意性（P値）とPRのオッズの比として定義される遺伝的影響の大きさ（オッズ比）によって、SNP-表現型の関係をフィルタリングすることができますSNP対立遺伝子のない個人における疾患の存在のオッズ以上の特定の遺伝子型（SNP対立遺伝子）を有する個体における疾患のesence。 図6は、0.000001の0.05にP-値の範囲内の関連タブ下で濾過した結果を示しています。 図7は、我々が研究で見つかった新しい指標に基づいて、「喘息」のための特異的な薬物再利用の仮説を示しています。薬と病気の用語の共起を含む5 Medlineの抄録の最小数でフィルタリングするための潜在的なタブの下で、図8に示す結果。この例では、遺伝子タブの下にあるすべての薬の結果は、元の問合せ語句（ 図9）に対応する、「IL2RB」遺伝子を対象としています。 IL2RB遺伝子上のアゴニストとして作用するすべての薬剤を返すために、「アクション」タブの下で濾過最後に、 図 10の結果を示します。 <img alt="図1"SRC = &quot;/ファイル/ ftp_upload / 54948 / 54948fig1.jpg&quot; /&gt; 図1：RE： 罰金薬インタラクティブなダッシュボードのホームページ。ユーザーは、薬剤名、新しい病気の兆候や遺伝子記号を入力することで、クエリを開始することができます。リンクはまた、薬物再利用の仮説を生成するための方法を記述GWASとPheWAS基準紙のために提供されます。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 図2： クエリのエントリの自動入力機能。例として、遺伝子の照会用語「IL2RB」が自動的遺伝子用語として認識されました。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <pキープtogether.withinページ= &quot;1&quot;&gt;：FOクラス= &quot;jove_content&quot; <img alt="図3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 図3： 遺伝子ベースのクエリ（ 例えば、IL2RB）から製造された医薬品転用結果テーブル。 IL2RB遺伝子について十二薬剤転用仮説が生成されます。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 図4：結果ページから個々の薬剤のための情報ページ。 「情報」列からアイコンをクリックすると、薬物ダクリズマブの詳細情報を示しています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図5： 薬物タブの下で高度な検索オプションは、特定の薬物によってフィルタリングします。この例では、3つの結果は、薬物ダクリズマブについて示されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 図6：DrugBank データベースから特定の疾患指標によってフィルタリングする薬剤タブの下で高度な検索オプション。病気の用語「移植」するための既知の適応症を持つすべての薬が示されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図7：アソシエーション]タブの下で高度な検索オプションは、有意水準によってフィルタリングします。この場合には、8個の結果は、その関連有意水準は0.05 0.000001のp値の範囲内に設けられています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 図8： 病気]タブの下で高度な検索オプションは、我々はこの研究で抽出された特定の疾患の徴候によってフィルタリングします。この例では、4つの結果は、新しい使用疾患適応症として喘息のために示されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">より大きなversiを表示するには、こちらをクリックしてください。</a>この図の上。 <img alt="図9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 図9：Medline の抄録での薬物および疾患用語の共起によってフィルタリングするための潜在的なタブの下で高度な検索オプション。この例では、4つの結果は、薬物や病気の用語の共起を含む5 Medlineの抄録の最小数によってサポートされていることが示されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 図10： 遺伝子タブの下で高度な検索オプションは、すべての結果が元のクエリに使用IL2RB遺伝子に対応する特定の遺伝子記号によってフィルタリングします。この例では、すべての薬物結果遺伝子タブの下で、元の問合せ語句に対応する、「IL2RB」遺伝子を対象としています。唯一のアゴニスト薬によってフィルタリングするアクション]タブの[詳細検索オプション。この例では、6つの結果がIL2RB遺伝子上のアゴニストとして作用するすべての薬剤に対して戻されます。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

薬物転用仮説生成の使用」RE：罰金薬「システム

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

drug-repurposing-hypothesis-generation-using-the-refine-drugs-system

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.2K ビュー.  The Ohio State University. Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options. 

ビデオ: Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

生化学

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

ゼブラフィッシュで自動化されたハイコンテンツ炎症アッセイ：創薬を促進する

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

免疫・感染学

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

創薬における多能性幹細胞由来肝細胞様細胞を誘導人間を使用して

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

発生生物学

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

NMRベースのフラグメントスクリーニングは、最小サンプルで最大限の自動化モードで

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

フラグメントベースのリード発見におけるスクリーニングのためのナノ差動走査フルオリメトリー

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

の実装<em&gt;インビトロ</em&gt;薬剤耐性アッセイ：臨床的に関連性のメカニズムを暴くための可能性を最大化

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

医学

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

バイオセンサーベースの高スループットバイオパンニングおよびバイオインフォマティクス分析戦略による、薬物-タンパク質相互作用のグローバル検証

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

生物工学

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

タンパク質 - タンパク質相互作用の阻害剤の開発を通じてREPLACE：アプリケーションの設計と開発非ATP競合CDK阻害剤に

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

生物学

Chemogenetic Regulation in Reprogrammed Stem Cell-derived Precursor Cells in Treating Neurodegenerative Diseases

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for precise neuronal modulation. Here, we utilized CRISPR-engineered human reprogrammed stem cells expressing excitatory (hM3Dq) or inhibitory (hM4Di) DREADD receptors to evaluate the functional integration and modulation of transplanted dopaminergic precursors in a murine model of Parkinson&#39;s disease (PD). Key steps included generating non-fusion DREADD-expressing stem cell lines, differentiating them into midbrain dopaminergic precursors, and transplanting these cells into the striatum of 6-hydroxydopamine (6-OHDA)-lesioned mice. We conducted behavioral assessments and electrophysiological recordings to analyze the effects of the transplanted cells. Behavioral tests, such as the cylinder test, demonstrated significant modulation of motor function following clozapine-N-oxide (CNO) administration. Specifically, activation of hM4Di reduced contralateral forelimb movement, whereas activation of hM3Dq was associated with enhanced motor behavior. Electrophysiological recordings revealed distinct synaptic responses. hM4Di activation increased interevent intervals and decreased peak amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs), whereas hM3Dq activation decreased interevent intervals and increased peak amplitudes, reflecting enhanced excitatory signaling. In summary, the integration of behavioral and electrophysiological assessments validates the precise functional incorporation of engineered chemically reprogrammed stem cells into host neural circuits.

Here, we describe a protocol for engineering chemically reprogrammed stem cells to achieve precise neuronal modulation by differentiating these cells into dopaminergic precursor cells, transplanting them into mouse models of Parkinson's disease, and evaluating behavioral and electrophysiological outcomes to confirm the successful integration and functional effectiveness of the transplanted cells.

神経変性疾患の治療における再プログラム幹細胞由来前駆細胞における化学遺伝学的制御

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for ...

神経科学

Drug Discovery: Overview

Drug discovery is a multifaceted process involving extensive screening, testing, and optimization of lead compounds to identify potential new drugs for therapeutic use. It combines several approaches, including screening large numbers of natural products, chemical modification of known active molecules, identification of new drug targets, and rational design based on biological mechanisms and drug-receptor structure. These approaches are carried out in both academic research laboratories and the pharmaceutical industry.
The process begins with drug screening, which involves various assays to evaluate the pharmacological profile and activity of the drug at different levels, such as molecular, cellular, organ system, and whole animal levels. The candidate molecule undergoes extensive testing to determine its mechanism of action, selectivity, and potential toxic effects. Animal models of human diseases are used to select compounds for further development, especially in cases where preclinical models are well-established.
The candidate compound is screened for its activity on the target protein at the molecular level using techniques like receptor binding assays. Cell function studies determine whether the drug acts as an agonist, partial agonist, inverse agonist, or antagonist at relevant receptors. Whole animal studies assess the drug&#39;s effects on organ systems and disease models.
Lead compounds with the desired pharmacological activity and selectivity are identified during the screening and lead generation processes. These lead compounds serve as the basis for further chemical modification and optimization in the lead optimization phase. Lead optimization involves a range of tests and assays to increase the potency of the compound, optimize selectivity, and evaluate other characteristics, such as pharmacokinetic properties. Animal studies assess the compound&#39;s activity in vivo, potential adverse effects, genotoxicity, and oral availability.
Only a small percentage of drug discovery projects succeed in generating a drug candidate, and the lead optimization phase can be time-consuming and challenging. Many factors can hinder progress, including the inability to optimize lead compounds or the failure to produce desired effects in animal models. Successful drug candidates proceed to the next phase, preclinical development, before entering human clinical trials.

創薬:概要

Drug discovery is a multifaceted process involving extensive screening, testing, and optimization of lead compounds to identify potential new drugs for ...