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. 유전자에서 쿼리의 개시, 약물 또는 질병 약관 <ol><li> http://drug-repurposing.nationwidechildrens.org : 다음 링크에서 &quot;좋은 약 RE&quot;의 홈페이지에 액세스 할 수 있습니다. 의약품 (제네릭 의약품 명), 질병 (새 질환 표시) 또는 유전자 (공식 HGNC 유전자 기호) : 다음과 같은 세 가지 범주 중 하나에서 검색 창에 검색어를 입력하여 시작합니다. </li><li> 전용 &quot;약물 이름&quot;, &quot;새로운 질병의 표시&quot;, &quot;유전자 기호&quot;또는 &quot;전체&quot;카테고리를 포함하도록 검색 창 기능을 필터링합니다. 검색 창 쿼리 항목에 대한 자동 채우기 기능을 포함하고 있습니다. </li><li> 키워드에 넣고 &quot;검색&quot;버튼을 클릭합니다. 정렬 다음 열 중 하나에 의해 결과의 테이블 : &quot;약&quot;, &quot;등록 표시&quot;, &quot;P-값&quot;, &quot;P-값이 조정&quot;, &quot;확율 비율&quot;, &quot;연구&quot;, &quot;새로운 표시&quot;, &quot;의약품 은행 표시 &quot;,&quot;Medline을 초록의 # &quot;,&quot;CLI의 #nical 시험 등록 &quot;,&quot;잠재적 인 &quot;,&quot;SNP &quot;,&quot;유전자 &quot;또는&quot;작용 &quot;. </li><li> 약물 정보 기능을 활성화하기 위해 고급 검색 옵션으로 이동합니다. 특정 약물에 대한 &#39;정보&#39;항목에서 아이콘을 클릭합니다. 나열하는 페이지를 준수합니다 모든 협회, 질병 이름, 약 이름에 대한 P 값, 유전자 정보 (NCBI 유전자 링크) 의약품 정보 (DrugBank 링크)를 포함하여 해당 정보를 제공합니다. </li></ol> 고급 옵션 2. 탐험 <ol><li> 페이지의 오른쪽에있는 &quot;고급&quot;버튼을 클릭하고, 또한 결과를 수정하는 몇 가지 옵션이 제공됩니다. 약물, 협회, 질병, 잠재력, 유전자 및 조치 : 고급 검색 옵션은 다음에 대한 수정을 포함한다. </li><li> 수출은 페이지의 오른쪽에있는 &#39;내보내기&#39;버튼을 클릭하여 테이블을 발생합니다. 고급 검색 창을 아래로 접어하기 위해서 &quot;단순&quot;버튼을 클릭합니다. &lt;/ 리&gt; <li> 고급 옵션 &quot;약물&quot;탭에서, 특정 약물 표시 또는 결과를 필터링하는 추가 약물의 이름을 지정합니다. </li><li> 은 &quot;연결&quot;탭에서, 유의 수준의 P-값, FDR과 조정 된 P-가치, 효과 크기 (확율 비율) 및 / 또는하여 필터 결과 유형 (GWAS, PheWAS 또는 둘 다)을 공부한다. </li><li> 은 &quot;질병&quot;탭에서 예측 된 새로운 사용하기위한 특정 질병에 대한 설명을 지정합니다. </li><li> 은 &quot;전위&quot;탭에서 다음 조건 중 어느 한 항에있어서, 필터 결과는 (i) 약물 표시가 DrugBank 데이터베이스에 포함되어 있는지 여부, 약물의 동시 발생 및 질병과 Medline을 초록의 (II)의 수 ( 약물의 동시 발생과 질병 (IV) 잠재적 용도 변경과 ClinicalTrials.gov 데이터베이스 항목의 ⅲ) 수. 주 : (II)를 강하게지지 관계가 이미 DrugBank 데이터베이스에 존재 : 알려진 (I) 다음 잠재적 용도 변경 옵션은 검색의 신규성을 설명: 두 임상 시험 레지스트리와 Medline을 초록의 일부 지원, (ⅲ) 가능성 : 임상 시험 레지스트리 또는 Medline을 초록과 중 일부 지원 (ⅳ) 소설 : 임상 시험 레지스트리도 Medline을 초록의 증거. </li><li> 은 &quot;유전자&quot;탭 아래에서 SNP ID를 입력하거나 유전자 기호는 특정 약물 표적 유전자에 의한 결과를 필터링 할 수 있습니다. </li><li> &quot;작업&quot;탭에서 작용제, 길항제, 기타, 알 수없는 또는 모든 (: DrugBank 데이터베이스 소스)를 포함하는 약물 표적 (들)에 대한 약물 작용의 유형을 지정합니다. </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.

높은 처리량 약물 및 납 화합물 스크리닝을 포함하여 기존의 신약 개발 접근 방식과 관련된 비용이 많이 들고 비효율적 인 프로세스는, 환자 1, 2에 대한 치료에 대한 연구의 발견을 번역에 지연에 기여하고있다. 10 억 달러로 15 ~ 20 년 평균은 침대 옆 3 벤치에서 새로운 약을 가지고해야합니다. 또한, 약물의 52 %는 1 상 임상 시험에서 개발하는 동안 실패하고, 2 단계를 입력 화합물의 25 %는 전체 3 상 임상 시험 (4)에 진행합니다. 약물의 용도 변경 또는 약물 재배치의 목적은 실패 마약을 갱신 및 / 또는 더 빨리 환자들에게 새로운 치료법을 제공하고 높은 성공률과 함께하기 위해 승인 된 의약품에 대한 새로운 적응증을 찾는 것입니다. 의약품의 용도 변경은 3-12년 5 환자에 사용되는 약물을 사용할 수 있도록 타임 라인을 감소시킬 수있다. 약물 용도 변경을위한 중요한 의료 응용 프로그램은 다음과 같습니다 가난한 prognos과 질병하고 낮은 생존율, 약제 내성 질병, 충분한 자금 질환 연구 분야와 가난하고 소외된 환자 집단. 전산 약물의 용도 변경은 설계 및 약물 후보 6 새로운 적응증에 대한 가설을 생성 할 수있는 자동화 된 워크 플로우를 검증하는 과정으로 정의된다. 기존 컴퓨팅 약제 용도 변경 방법은 타겟 기반 기술 기반 서명 기반, 네트워크 기반의 분류 및 타겟팅 메커니즘 - 기반 및 유전자 질환 또는 약물 관점에서 배향 될 수있다. 또한, 계산 방법은 또한 용도 변경 약물 후보 7 개념 증명 검증 실험 및 소규모 임상 연구를 가속화 할 수 있습니다. 우리는 이전에 &quot;RE : 좋은 약&quot;에보고 한 약물 유전자 - 질병의 관계 (8)의 전이 이론을 바탕으로 의약품 용도 변경 가설 생성을위한 자유롭게 사용할 대화 형 웹 기반 도구입니다. 전체 g이 방법의 OAL 체계적으로 임상, 산업 및 규제 지역 사회 등 다양한 지역 사회에서 사용자의 용도 변경 약물을 사용하는 약물, 유전자 및 임상 데이터의 다양한 유형을 통합하는 것이다. 이 시스템에 대한 기초적인 방법은 이전 게놈 넓은 협회 연구 (GWAS) 연구 9,10 용도 변경 약물에 발 형체 전체 연관 연구 (PheWAS) 데이터의 사용에 대한보고되었다. 이러한 유형의 데이터의 새로운 조합은 다른 목표 기반의 방법 6,11에서 우리의 웹툴을 구분합니다. 재 : 고급 마약 시스템은 현재 916 마약, 567 유전자와 1,770 질병을 다루는 60,911 약물 용도 변경 가설이 포함되어 있습니다. 웹툴 대화 형 약물 용도 변경 가설을 검색하고 다양한 기준을 사용하여 우선 순위를 연구자를위한 사용자 친화적 인 인터페이스를 제공합니다. 예를 들어, 사용자는 생물 의학 문헌에서 지원 및 임상 시험 databa와 약물 용도 변경 가설을 필터링 할 수 있습니다그 자체로 상당한 P-값, 협회 확률 비율 또는 특정 표시로. 이 시스템에 대한 유일한 요건은 인터넷이다.

<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.

이 예에서, 유전자 &quot;IL2RB&quot;는 유전자 기반 쿼리로 입력하고, 자동으로 자동 완성 기능 (도 1)에 의해 그렇게 인식 하였다. 도 2에 도시 된 바와 같이 IL2RB 유전자의 열두 약제 용도 변경 가설이 반환된다. 이 경우 특정 약물의 용도 변경 가설, &quot;클리&quot;에 대한 상세 정보 페이지는 &quot;정보&quot;열 (그림 3)에서 제공된다. 약물 탭은 &quot;클리&quot;약물에 대응하는 모든 결과가되었다에도 4에 도시 된 바와 같이 그 결과를 여과 하였다. 그림 5는 &quot;이식&quot;질병 용어 (: DrugBank 데이터베이스 소스)에 대한 알려진 표시 만 그 약을 보여줍니다. 협회 탭 홍보 확률의 비율로 정의 통계적 유의성 (P-값) 유전 효과 크기 (교차비)에 의해 SNP-표현형 관계를 필터링 할 수있는 사용자를 할 수 있습니다염기 다형성 대립 유전자가없는 개인 질병의 존재의 가능성을 통해 특정 유전자형 (SNP 대립 유전자)와 개인의 질병의 esence. 도 6은 0.000001 0.05로 P 값의 범위 내에서 연결 탭 필터링 된 결과를 나타낸다. 그림 7은 우리가 연구에서 발견 된 새로운 적응증에 따라 &quot;천식&quot;에 대한 특정 약물 용도 변경 가설을 보여줍니다. 잠재적 탭에서 그림 8은 결과 약물과 질병 용어의 동시 발생을 포함하는 5 Medline을 초록의 최소 숫자로 필터링합니다. 이 예에서, 유전자 탭 모든 약물 결과를 원래의 질의 용어 (도 9)에 대응하는 &quot;IL2RB&quot;유전자를 대상으로한다. 마지막으로, &quot;작업&quot;탭에서 여과 그림 10은 결과는 IL2RB 유전자에 작용 물질의 역할을 모두 약물을 반환합니다. <img alt="그림 1"SRC = &quot;/ 파일 / ftp_upload / 54948 / 54948fig1.jpg&quot;/&gt; 그림 1 : RE : 좋은 마약 대화 형 대시 보드 홈페이지. 사용자는 약 이름, 새로운 질병의 표시 또는 유전자 기호를 입력하여 조회를 시작할 수 있습니다. 링크는 또한 약물의 용도 변경 가설을 생성하는 방법을 설명하는 GWAS와 PheWAS 참조 서류 제공됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 그림 2 : 쿼리 항목에 대한 자동 채우기 기능. 예로서, 유전자 검색어 &quot;IL2RB&quot;가 자동 유전자 용어로 인식되었다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <p유지-together.within 페이지 = &quot;1&quot;&gt; : FO 클래스 = &quot;jove_content&quot; <img alt="그림 3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 그림 3 : 유전자 기반 쿼리 (예를 들어, IL2RB)에서 생산되는 의약품 용도 변경 결과를 테이블. IL2RB 유전자에 대한 십이 약물 용도 변경 가설이 생성된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 그림 4 : 결과 페이지에서 개별 의약품에 대한 정보 페이지. &#39;정보&#39;항목에서 아이콘을 클릭하면 약물 클리에 대한 상세한 정보를 보여줍니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 5 : 약물 탭에서 고급 검색 옵션은 특정 약물에 의해 필터링합니다. 이러한 예에서, 세 가지 결과는 약물 클리 위해 도시된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 그림 6 : 약 탭에서 고급 검색 옵션은 DrugBank 데이터베이스에서 특정 질병의 지시에 의해 필터링합니다. 질병 용어는 &quot;이식&quot;에 대한 알려진 표시 모든 약물이 표시됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 7 : 연결 탭에서 고급 검색 옵션은 유의 수준을 기준으로 필터링합니다. 이 경우, 팔의 연관 결과 유의 수준 0.05-0.000001의 P 값의 범위 내에 설치되어있다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 그림 8 : 질병 탭에서 고급 검색 옵션은 우리가이 연구에서 추출 된 특정 질병의 지시에 의해 필터링합니다. 이 예에서, 네 개의 결과 새로운 용도 질환 표시 천식에 대해 도시된다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">큰 versi을 보려면 여기를 클릭하십시오</a>이 그림의에. <img alt="그림 9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 그림 9 : 잠재적 탭에서 고급 검색 옵션은 Medline을 초록 약물과 질병 용어의 동시 발생에 의해 필터링합니다. 이 예에서, 네 개의 결과 약물 질병 용어의 동시 발생을 함유하는 5 Medline을 초록의 최소 수에 의해 지원되는 것을 나타낸다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 그림 10 : 유전자 탭에서 고급 검색 옵션은 모든 결과는 원래의 질의에 사용되는 IL2RB 유전자에 해당하는 특정 유전자 기호에 의해 필터링합니다. 이 예에서, 모든 약물 결과유전자 탭에서 원래 검색어에 해당하는 &quot;IL2RB&quot;유전자를 대상으로하고 있습니다. 작업 탭에서 고급 검색 옵션 효능 약물에 의해서만 필터링합니다. 이 예에서, 여섯 결과는 IL2RB 유전자에 작용 물질의 역할을 모든 의약품에 대한 반환됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

은 &quot;RE를 : 좋은 약&quot;을 사용하여 의약품 용도 변경 가설 발전 시스템

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 조회수.  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. 

비디오: 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.

효율적인 Refolding 및 Chemoreceptor Ligand 바인딩 도메인의 정화 방법

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

연구

생화학

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.

촉진 약물 디스커버리 : Zebrafish의 자동화된 하이 컨텐츠 염증 분석

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

면역학 및 감염병학

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.

약물 발견에 대 한 만능 줄기 세포 유래 Hepatocyte 같은 세포를 유도 하는 인간을 사용 하 여

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

발생학

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.

최소 샘플이지만 최대 자동화 모드에서 NMR 기반 단편 스크리닝

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.

단편 기반 리드 디스커버리에서 스크리닝을 위한 나노 차분 스캔 형광

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.

구현<em&gt; 체외</em&gt; 약물 저항 분석법 : 임상 적 저항 메커니즘을 폭로 가능성을 극대화

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

의학

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.

약물 단백질 상호 작용의 글로벌 검증을 위한 바이오센서 기반 고처리량 바이오패닝 및 생물정보학 분석 전략

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

생체공학

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.

를 통해 교체 단백질 - 단백질 상호 작용의 억제제의 개발 : 설계 및 개발 비 ATP 경쟁 CDK 억제제에 응용

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

생물학

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.

신경퇴행성 질환 치료에서 재프로그래밍된 줄기세포 유래 전구세포의 화학유전학적 조절

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

신경과학

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.

Drug Discovery: 개요

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

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