Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

주의 : 사용하기 전에 모든 관련 화학 물질의 안전 보건 자료 (SDS)를 참조하시기 바랍니다. 약물 스크리닝 실험은 대량의 대응에 비해 추가 위험이있을 수 있습니다 나노 물질의 합성 및 처리를 포함한다. 확인하십시오 모든 필요한 통제 조치는 엔지니어링 컨트롤 (흄 후드) 및 개인 보호 장비 (PPE, 예를 들어, 안전 길이 바지, 폐쇄 발가락 신발, 내 화학성 장갑, 안전 고글)의 사용을 포함, 실험을하는 동안 연습을한다. 약물 검사 용 화학 시약 1. 준비 <ol><li> 금 나노 합성 전구체 <ol><li> 금 (III) 클로라이드 용액을 15 mm로 제조 초순수 6.9 ㎖에 금 (III) 클로라이드 용액 (99.99 % 미량 금속 기준으로, 묽은 염산 30 중량. %) 30 mg을 용해. 주의 : 금 염화물 용액은 부식성과 자극성이다. 눈과 피부에 직접 접촉을 방지하기 위해 적절한 개인 보호구를 착용 할 것. </li><li> 디초순수 1 ㎖의 HSA 또는 BSA의 ssolve 74 mg을 74 밀리그램 / 단백질 원액 ml의 준비를한다. </li></ol></li><li> 약액 <ol><li> 목적하는 약물의 양, 예를 들면, (a) 이부프로펜 1.9 ㎎, (b) 와파린 2.8 ㎎, (c) 페니토인 2.3 ㎎, 450 mm의 제조 DMSO 20 μL의 (d) 술 파닐 아미드 1.5 ㎎을 용해 약물 재고 솔루션. 주 : 이들 약물은 소수성이고 물에 난 용성이 때문에 DMSO 용매로서 선택된다. </li></ol></li><li> 기타 시약 <ol><li> NaOH 용액 1.5 M을 제조 초순수 10ml에 수산화 나트륨 펠렛을 600 mg을 녹인다. 우레아 수용액의 20 M을 제조 초순수 2 ㎖에 우레아 2.4 g을 용해. </li></ol></li></ol> 단백질 템플릿 형태 금 나노 2. 합성 <ol><li> HSA-주형 금 나노 (HSA-금 나노) <ol><li> 두 개의 개별 온도 제어 자기 교반에 두 개의 유리 병, 거기에 마이크로 자기 교반 막대를 포함하는 각을 배치rers. </li><li> 60 ° C로 한 마그네틱 스터 러의 온도를 설정하고, &quot;60 ℃&quot;로 그 위에 유리 바이알 라벨; 다른 자기 교반기는 그 위에 유리 바이알 &quot;RT&quot;로 표시되는 상온 (RT)에서 유지된다. </li><li> 단백질 주형 내부 금 이온의 밀봉을 허용하도록 HSA 용액 200 μL, 초순수 200 μL, 및 (달리 명시하지 않는 한 360 RPM으로 설정) 일정한 교반 하에서 각각의 바이알에 금 (III) 클로라이드 용액 200 μL를 추가한다. </li><li> 금 나노 결정을 형성 HSA의 환원 능력을 활성화하고 0 분으로 반응 시간을 기록하기 시작하도록 2 분 후에, 각각의 바이알에 NaOH 용액 20 μL를 추가한다. </li><li> 매 20 분, 384 웰 검은 판에 샘플의 각각의 솔루션의 50 μl를 그리는 마이크로 플레이트 리더와 발광 스펙트럼을 측정한다. 일반적인 스캔 설정 : - 850 nm의 λ 전 = 370 nm의은, λ EM은 410 =. </li><li> 100 분 후 자석 교반기를 중지합니다. </li><li> 싱크대에 흐르는 물에 유리 병을 쿨. 주의 : 유리 병이 뜨겁습니다. 연소를 방지하기 위해 내열 장갑을 착용 할 것. </li><li> 각 샘플은 다른 온도 조건에서의 Au 나노 결정의 형성이 반응 속도를 획득하기 위해 모든 시점에서 광전자 스펙트럼 플롯. </li></ol></li><li> BSA-주형 금 나노 (BSA-금 나노) <ol><li> 자기 교반기 위에 마이크로 자기 교반 막대를 함유하는 유리 바이알을 놓는다. 실내 온도와 같은 온도를 남겨주세요. </li><li> BSA 용액 200 μL, 우레아 200 μL, 일정한 교반하에 유리 바이알에 금 (III) 클로라이드 용액 200 μL를 혼합한다. </li><li> 2 분 후에, 금 나노 결정을 형성하기 위해 BSA의 환원 능력을 활성화하고 0 분으로 반응 시간을 기록하기 시작하는 NaOH 용액 20 μL를 추가한다. 시간당 반응 혼합물의 광전자 스펙트럼을 측정한다. 주 : BSA - 금 나노 결정의 광전자 스펙트럼을 측정덜 빈번 BSA - 금 나노 결정의 형성 비율 인해 우레아 이하의 효과를 동일 온도에서 HSA-Au로보다 느리게하기 때문에 단백질을 전개한다. </li><li> 7 시간 후 자석 교반기를 중지합니다. 요소의 변성에 의해 금 나노 결정의 형성 반응 속도를 볼 수있는 모든 시간에 광전자 스펙트럼을 플롯. </li></ol></li></ol> 3. 작은 분자 약물 검사 <ol><li> HSA에 다른 작은 분자 약물의 화면 상대 결합력 <ol><li> 다섯 유리 병, 온도 제어 멀티 자석 교반기 위에 거기에 자기 교반 막대를 포함하는 각 놓습니다. A, B, C로이 병에 레이블을 각 약물, 즉 이부프로펜, 와파린, 페니토인, 그리고 설파 닐 아미드에 대한 각각 거라고. 제어 순수한 HSA와 함께 한 레이블. </li><li> 각 바이알에 HSA 200 μl를 추가합니다. 네 대응하는 유리 병에 약액의 1 μl를 추가합니다. 컨트롤에 DMSO의 1 μl를 추가합니다. 교반기 전환하고 360 회전 속도를 설정RPM 및 HSA에 결합하는 약물을 완료 할 수 있도록 1 시간 동안 품어. </li><li> 1 시간 후, 밀리 Q 물 200 μL와 일정한 교반하면서 각각의 유리 병에 금 (III) 클로라이드 용액 200 μl를 추가합니다. 10 분 동안 일정하게 교반하면서 60 ℃로 온도를 설정합니다. 각 바이알에 1.5 M NaOH를 20 μl를 첨가하고 0 분으로 반응 시간을 기록하기 시작한다. </li><li> 신속하게 방출 스펙트럼을 (결과는 0 분으로 표시) 384 웰 블랙 플레이트에 각 바이알에서 용액 50 μL를 그리고 측정한다. 각 샘플마다 10 분의 발광 스펙트럼을 녹음. 네 개의 다른 시간에 적어도 네 개의 스펙트럼을 수집, 즉, 0, 10, 20, 및 30 분이다. </li><li> 일관된 추세에 결과를 얻기 위해 3.1.4 여러 번 - 반복 3.1.1 단계를 반복합니다. 자석 교반기를 중지합니다. </li><li> (- D 및 제어) 각 샘플에 대한 시간 - 분해 광전자 스펙트럼 플롯. 다이에서의 Au 나노 결정의 형성 반응 속도를 비교하는 시간에 대한 모든 샘플 플롯의 피크 강도를 파악fferent 약물로드 단백질 템플릿. </li></ol></li><li> HSA에 특정 약물의 결합 상수를 측정 <ol><li> 3.1.4 - 반복 3.1.1 단계를 반복합니다. (단 약물 로딩없이 HSA를 사용하여 제어 샘플 포함) 네 가지 농도에서 이부프로펜 솔루션 약물 솔루션을 교체합니다. 대응 약물 농도에 따른 유리 바이알에 라벨. </li><li> 10 분 후, 싱크대에서 흐르는 물에 유리 튜브를 냉각. 주의 : 유리 병이 뜨겁습니다. 연소를 방지하기 위해 내열 장갑을 착용 할 것. </li><li> 384 웰 검은 접시에 각각의 유리 병에서 위의 솔루션의 50 μL를 그리고, 발광 스펙트럼을 측정한다. </li><li> 다른 약물 농도 결과의 또 다른 두 세트를 얻기 위해 3.2.3 두 번 더 - 반복 3.2.1 단계를 반복합니다. </li><li> 자석 교반기를 중지합니다. 광전자 스펙트럼을 플롯 한 결과를 분석한다. <ol><li> 각각의 배치에서 얻어진 미가공 데이터를 위해, 각각의 광전자 스펙트럼 플롯소프트웨어 샘플 등 OriginPro의 소프트웨어와 같은 피크 강도를 찾을 수 있습니다. </li><li> 상대 형광 강도 계산 및 플롯 [(I 0 - I)을 / I 0] 나는 내가 각각 약물로드 HSA 순수한 HSA에 형성된 금 나노 결정의 형광 강도를 참조 0 약물 농도에 대하여. </li><li> 미카엘리스 - 멘텐 방정식을 사용하여 단일 부위 결합 모델에 데이터를 피팅함으로써 결합 상수를 계산한다 : R 최대가 최대 응답 신호가 나타내는 C / (K D + C) × Y = R 최대를, C는 리간드의 농도이고 D 및 K는 결합 상수이다. 이러한 OriginPro의 같은 소프트웨어에서이 작업을 수행합니다. 비선형 커브 피팅 피팅 메뉴 분석을 선택한 다음, 성장 / S 자형 범주에서 힐 기능을 선택합니다. 설정에서 : 기능 선택 페이지에서 장착 결과를 표시하는 맞춤을 클릭합니다. </li></ol></li></ol></li></ol>

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Y., Li, H. W., Liu, Z. Y., Wu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-Assisted+Synthesis+of+BSA-Protected+Small+Gold+Nanoclusters+and+Their+Fluorescence-Enhanced+Sensing+of+Silver(I)+Ions.">Microwave-Assisted Synthesis of BSA-Protected Small Gold Nanoclusters and Their Fluorescence-Enhanced Sensing of Silver(I) Ions.</a> Nanoscal. 4, 2251-2254 (2012).</li><li>Liu, Y., Ai, K., Cheng, X., Huo, L., Lu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanocluster+Based+Fluorescent+Sensors+for+Highly+Sensitive+and+Selective+Detection+of+Cyanide+in+Water.">Gold Nanocluster Based Fluorescent Sensors for Highly Sensitive and Selective Detection of Cyanide in Water.</a> 20, 951-1907 (2010).</li><li>Liu, J., Yu, M., Zhou, C., Yang, S., Ning, X., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+Tumor+Targeting+of+Renal-Clearable+Luminescent+Gold+Nanoparticles:+Long+Tumor+Retention+and+Fast+Normal+Tissue+Clearance.">Passive Tumor Targeting of Renal-Clearable Luminescent Gold Nanoparticles: Long Tumor Retention and Fast Normal Tissue Clearance.</a> J. Am. Chem. Soc. 135, 4978-4981 (2013).</li><li>Negishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Loading+of+Small+Aun+Clusters+(n+=+10&#8211;39)+onto+BaLa4Ti4O15+Photocatalysts:+Toward+an+Understanding+of+Size+Effect+of+Co-Catalyst+on+Water+Splitting+Photocatalytic+Activity.">Controlled Loading of Small Aun Clusters (n = 10&#8211;39) onto BaLa4Ti4O15 Photocatalysts: Toward an Understanding of Size Effect of Co-Catalyst on Water Splitting Photocatalytic Activity.</a> J. Phys. Chem. C. , (2015).</li><li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoclusters.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.</a> J. Am. Chem. Soc. 131, 888-889 (2009).</li><li>Celej, M. 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The authors have nothing to disclose.

이 방법으로 강조 할 몇 가지 중요한 단계가 있습니다. 다른 작은 분자 약물의 상대적 결합 친 화성을 심사의 프로토콜이 단계에서 3.1.2, 3.1.3 및 3.1.4은 상대 바인딩 강도 일관된 추세를 보여주는 좋은 결과를 얻을 중요하다. 이 단계에서, 측정 용 반응 액을 화학 물질을 첨가하여 드로잉 작업은 시간 지연 효과 및 일관성을 보장하기 위해 실시한다 측정 반응 액을 화학 물질을 첨가 및 도면의 동일?...

인간 혈청 알부민 (HSA)과 소 혈청 알부민 (BSA)와 같은 혈청 알부민은 혈장에서 가장 풍부한 단백질이고 혈액 구획의 삼투압을 유지하는데 중요한 역할을한다. 또한 스테로이드와 같은 낮은 수용해도의 소분자, 지방산, 갑상선 호르몬, 약물의 광범위한 담체 단백질로서 인식된다. 바인딩 재산권 알부민 혈청하는 이들 분자의 (예를 들어, 친 화성 또는 강도를 결합, 결합 부위)의 약동학에 중요한 주제를 형성한다. 1-4 여러 분석 방법과 같은, 알부민 혈청 상이한 약물의 결합 특성을 연구하기 위해 개발되어왔다 X 선 결정학, 5,6- 핵 자기 공명 (NMR), 7-11 및 표면 플라즈몬 공명 (SPR), 12, 13 등 그러나, 이들 방법은 지루하고 시간 소모적 해석 처리 (하나에 의해 제한되는 예를 들면, X 선 crystallo위한 단결정 성장그래픽 연구), 전문 및 고가의 장비의 요구 사항 (SPR), 또는 검출을위한 비용이 많이 드는 동위 원소 표지 (NMR)을 필요로한다. 또한, 빨리 감기 직쇄, 비용 효율적인 방법으로 저분자 약물 스크리닝하는 다른 방법을 개발하는 것이 매우 바람직하다. 금 나노 클러스터 (금 나노)는 그들의 분리 및 크기에 의존하는 전자 구조, (18)에 그들은 광범위한 연구 관심을 받고있다 2 나노 미터. 14 ~ 17보다 작은 크기의 금속 원자의 수십 포함 된 나노 물질의 특별한 유형이다 20-23 이러한 독특한 재료의 특성, 특히 강한 형광이, 생물학적 시스템에서 감지 및 영상. 24-32 초소형 형광 금 나노 기능성 단백질을 사용하여 합성 할 수있는 등 다양한 응용 프로그램을 발견했다. (19)와 분자 등의 흡수 및 배출, 이러한 주형으로서 혈청 알부민 등. (33)의 전형적인 주형 단백질 합성<html> 금 나노 결정은, 금 염 일정량 제 단백질 안에 캡슐화되고이어서 단백질 자체에 의해 감소​​된다. 단백질의 환원 능력은 알칼리성 용액의 pH를 증가시킴으로써 활성화 될 수있는 기능성의 아미노산 잔기 (예를 들어, 티로신) 성분에 기인한다. 단백질 구조의 전개는 금 나노 결정의 형성을위한 중요한 단계로 고려된다. 전개 된 단백질, 더 환원 관능기 캡슐화 금 염에 노출 될 수 있기 때문이다. 단백질 변성제에 열처리 또는 노광에 의해 달성 될 수 흘러 가고. 작은 분자 약물의 도입은 또한 중간 변성 온도 및 전개의 엔탈피를 수정 즉 전개 과정에 영향을 미칠 수있다. (34, 35) 이러한 모든 요인의 효과를 차례로 형광 금 나노 결정의 형성 반응 속도에 의해 반사 될 수 있으며 각성 얻어진 금 나노 결정의 형광 강도. 36 e_content는 &quot;&gt;이 비디오는 높은 온도 (60 ℃)에서 약물 - 로딩 알부민 단백질의 Au 나노 결정을 합성 또는 변성제 (예를 들면, 우레아) 얻어진 금 나노 결정의. 형광 강도의 존재 하에서 약물의 스크리닝 방법을 보여 신호 판독이다. 먼저, 금 나노가 금 나노. 초의 형성 반응 속도에 영향을 미치는 단백질 (열처리 또는 변성제에 의해 유도) 흘러 가고 방법을 보여 60 ℃에서 또는 우레아의 존재하에 처리 된 HSA 및 BSA 템플릿에서 합성되어, 금 나노 결정은 다른 약제와 함께 미리로드 단백질 템플릿을 합성하고, 얻어진 금 나노 결정의 상대적 형광 농도의 약물 로딩 효과는 상대적인 결합 강도의 측정을 제공하는 연구된다. 마지막으로, Au로 NC 약물 선별 프로토콜을 위해 수정 될 고정 된 농도의 단백질에 사전로드 약물 함량을 변화시킴으로써 약물 - 단백질 결합 상수 (K D)의 정량적 측정.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

우리는 금 나노 결정에서 방출되는 차동 형광 신호에 기초하여 약물 - 로딩 단백질 내의 형광 금 나노 클러스터 (금 나노)를 형성함으로써 표적 단백질에 작은 약물 분자의 결합 친화도를 결정하기위한 새로운 약물 스크리닝 방법을 보여준다. 인간 혈청 알부민 (HSA)과 소 혈청 알부민 (BSA)과 같은 단백질은 알부민 단백질 모델로서 선택된다. 4 개의 작은 분자 약물 알부민 단백질에 다른 결합 친화도의 (예를 들어, 이부프로펜, 와파린, 페니토인 및 설파 닐 아미드)를 시험한다. 이는 변성 조건 (즉, 60 ° C 또는 우레아의 존재하에)하에 약물로드 알부민 단백질 내부 형광 금 나노 결정의 형성 비율은 (약물)없이 깨끗 단백질에 형성된 것보다 느리다는 것을 발견 하였다. 또한, 상기와 같은 나노 결정 형성의 형광 강도는 반비례 알부민이 단백질 약물의 결합 친화도에 상관 관계가 발견된다. 특히,금 나노 결정 형성 속도 느린 약물 - 단백질 결합 친 화성이 높은, 이렇게 얻어진 금 나노 낮은 형광 강도가 관찰된다. 얻어진 금 나노 결정의 형광 강도 테스트 따라서 다른 약물의 상대적인 결합 강도의 단순한 측정을 제공한다. 이 방법은 단순히 고정 된 단백질의 단백질 농도로 사전로드 약물 함량을 변화시킴으로써, 특정 약물 - 단백질 결합 상수 (K D)를 측정하기 위해 연장된다. 측정 결과는 다른 프레스하지만 더 복잡한 방법을 이용하여 얻어진 값과 잘 일치.

단백질의 반응성 작용기 (예를 들어, 티로신 잔기)가 캡슐화 된 금 이온을 감소시키고 이에 따라 금 나노 결정의 형성 비율을 가속 노출 될 수 있기 때문에, 전개 된 단백질들은 단백질 주형의 Au 나노 결정의 형성에 중요한 절차이다. 가열 및 외부 변성제는 단백질 전개 과정을 촉진하기 위해 두 가지 일반적인 방법이다. (1)는 모델 단백질로서 HSA를 사용하여, 금 나노 결정의 형?...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

단백질 표적 소분자 약물 검사를위한 신속하고 정량적적인 형광 방법

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

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Research

JoVE Journal

Bioengineering

9.9K 조회수.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

비디오: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Micropatterned 세포를 사용하여 약물의 영향 향상된 시각화 및 정량 분석

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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생체공학

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

높은 처리량 Antifungal 약물 검사를위한 칸디다 albicans Biofilm 칩 (CA BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

아데 닐 레이트 사이 클라 제의 약물에 의한 과민성 : 작은 분자와 siRNA의 심사 응용 프로그램에 대한 분석 최적화 기술과 소형화

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

바이오 메디컬 응용 프로그램에 대한 Bioinspired 나노 수산화 인회석의 빠른 혼합 준비

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

세포질 기계 화학 Epigenetic 한정자를 리디렉션하여 억압 유전자 전사

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

오픈 소스 마이크로플레이트 호환 조명 패널용 애플리케이션

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...