High Throughput Screening
A Story by Medicilon
Medicilon has strong capability in assay development and compound screening.Our screening capacity could be 200x384 well/week,or customize it per clients' needs.
Medicilon has strong capability in assay development and compound screening.Our screening capacity could be 200x384 well/week,or customize it per clients' needs. We work closely with partners such as Selleck,MCE and Tocris that provide compound libraries, and we have more than a library of 3000 compounds in-house. In vitro enzymatic assaysScreening cascade - inhibitors of small moleculesEnzymatic assaysMeasurement of substrate reduction (ATP, NAD/NADP, SAM, Acetyl-CoA, peptide, DNA, RNA, etc.) - Down assayMeasurement of products - Up assay Enzymatic assay - example 1HTRF assay-Homogeneous Time Resolved FluorescenceDonor=Eu and Tb Acceptor=XL665 Pros:(1)Highly specific, high signal/background ratio;(2) Highly stable fluorescence signal;(3)Homogeneous reaction, high throughput.Cons:(1)Cost for specific antibody is relatively high;(2)Specific antibodies need to be developed for specific target. Enzymatic assay - example 2ADP-Glo assayPros:(1) Highly sensitive;(2) Could detect enzymes other than kinases. Enzymatic assay - example 3Z’-Lyte assayPros:(1)High signal/background ratio, high throughput;(2) Low cost.Cons:(1)Could not detect enzymes with complex reaction mechanism;(2)Compounds with fluorescence itself will be hard to be evaluated;(3)False positive data will occur when testing proteases. Enzymatic assay development " key factors
In vitro cellular assaysPhenotypic assays:Cell proliferation, migration, invasion, apoptosis, necroptosis, autophagy, etc.Functional assays:Downstream signal like protein phosphorylation, second messanger(Ca2+, cAMP, IP3, cGMP).Protein-protein interaction(PPI) in cells:NanoBiT,NanoBRET,BiFC. Cellular assay development - cytotoxicity test1. Cell seeding densityCell viability >80%Seed cells in a plate with different cell densityCulture for 72 to 96 hours,CTG/CCK8 detection of cell viability.Select the density with CTG/CCK8 signal in a linear rangeWindow (Signal/background ratio)>2 2. Incubation timeSelect the optimized density and seed cellsIncubate cells/compounds for different time pointsDetect cell viability with CTG/CCK8The time point with a best assay window and data quality will be used for further experimentsWindow (Signal/background ratio) >2 3. DMSO toleranceDifferent doses of DMSO were incubated with cells for 72 to 96 hours, cell viability were tested with CTG methodNote: DMSO typically is less than 0.5% in cellular assays 4. Z’ and edge effectQC criteria: Z’>0.4, Window>2Usually edge wells will not be used for long term culture 5. Compound validationPositive compound should be 5-fold within reference data Plate layout:
High throughput screeningHigh throughput screening assay flow chart1. Assay optimization (Z’, assay window, DMSO tolerance, reference compound test)2. Pilot study with thousands of compounds will be run first to test the assay3.Orthogonal assay will also be set up to eliminate the false positive compounds4. Run HTS with tens of thousands of compounds5. Hits rate usually will be set to 0.1% to 0.5% High throughput screening - case studyPSMA:Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed high throughput PSMA enzymatic assays to identify new regulators of PSMA. Z-factor test and assay window test384 well plates were used for assay validation, data from four plates showed that Z-factor is higher than 0.5, and assay window is higher than 10-fold. HIT identificationTotally 100 x 384 well plates were used for screening of more than 30,000 compounds, and finished in 10 days, hits rate is about 0.8% when cut off was set to 50% inhibition of PSMA activity.
SummaryMedicilon has strong capability in assay development and compound screening. We work closely with partners such as Selleck, MCE and Tocris that provide compound libraries, and we have more than a library of 3000 compounds in-house. Our screening capacity could be 200x 384 well/week, or customize it per clients’ needs.
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MedicilonCambridge, MA
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Medicilon is an integrated contract research organization (CRO), providing comprehensive one-stop new drug R&D services for pharmaceutical enterprises and scientific research institutions around the w.. more..
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