Editing High-throughput screening (section)

== Techniques for increased throughput and efficiency ==

Unique distributions of compounds across one or many plates can be employed either to increase the number of assays per plate or to reduce the variance of assay results, or both. The simplifying assumption made in this approach is that any N compounds in the same well will not typically interact with each other, or the assay target, in a manner that fundamentally changes the ability of the assay to detect true hits.

For example, imagine a plate wherein compound A is in wells 1–2–3, compound B is in wells 2–3–4, and compound C is in wells 3–4–5. In an assay of this plate against a given target, a hit in wells 2, 3, and 4 would indicate that compound B is the most likely agent, while also providing three measurements of compound B's efficacy against the specified target. Commercial applications of this approach involve combinations in which no two compounds ever share more than one well, to reduce the (second-order) possibility of interference between pairs of compounds being screened.

===Recent advances===
Automation and low volume assay formats were leveraged by scientists at the NIH Chemical Genomics Center (NCGC) to develop quantitative HTS (qHTS), a paradigm to pharmacologically profile large chemical libraries through the generation of full concentration-response relationships for each compound.  With accompanying curve fitting and cheminformatics software qHTS data yields half maximal effective concentration (EC50), maximal response, [[Hill coefficient]] (nH) for the entire library enabling the assessment of nascent structure activity relationships (SAR).<ref name="ReferenceA">{{cite journal | title = Quantitative High-Throughput Screening (qHTS): A Titration-based Approach that Efficiently Identifies Biological Activities in Large Chemical Libraries. |author1 = Inglese, J. |author2 = Auld, D.S. |author3 = Jadhav, A. | author4 = Johnson, R.L. |author5 = Simeonov, A. |author6 = Yasgar, A.|author7 = Zheng, W. | author8 = Austin, C.P. | journal = Proc. Natl. Acad. Sci. USA |date = 2006 |volume = 103|issue=31|pages = 11473–11478 |pmid = 16864780| doi = 10.1073/pnas.0604348103 | pmc=1518803|bibcode = 2006PNAS..10311473I|doi-access = free }}</ref>

In March 2010, research was published demonstrating an HTS process allowing 1,000 times faster screening (100 million reactions in 10 hours) at 1-millionth the cost (using 10<sup>−7</sup> times the reagent volume) than conventional techniques using drop-based microfluidics.<ref name="ReferenceA"/> Drops of fluid separated by oil replace microplate wells and allow analysis and hit sorting while reagents are flowing through channels.

In 2010, researchers developed a silicon sheet of lenses that can be placed over microfluidic arrays to allow the fluorescence measurement of 64 different output channels simultaneously with a single camera.<ref>{{cite journal| year = 2010| title = High-throughput fluorescence detection using an integrated zone-plate array| journal = [[Lab on a Chip (journal)|Lab on a Chip]]| publisher = [[Royal Society of Chemistry]]| volume = 10| pmid = 20300671| issue = 7| pages = 852–856| doi = 10.1039/b923554j | last1 = Schonbrun| first1 = E| last2 = Abate| first2 = A. R| last3 = Steinvurzel| first3 = P. E| last4 = Weitz| first4 = D. A| last5 = Crozier| first5 = K. B| citeseerx = 10.1.1.662.8909}}
*{{cite press release |date=February 22, 2010 |title=Marriage of microfluidics and optics could advance lab-on-a-chip devices |website=ScienceDaily |url=https://www.sciencedaily.com/releases/2010/02/100216113905.htm}}</ref>  This process can analyze 200,000 drops per second.

In 2013, researchers have disclosed an approach with small molecules from plants. In general, it is essential to provide high-quality proof-of-concept validations early in the drug discovery process. Here technologies that enable the identification of potent, selective, and bioavailable chemical probes are of crucial interest, even if the resulting compounds require further optimization for development into a pharmaceutical product. Nuclear receptor RORα, a protein that has been targeted for more than a decade to identify potent and bioavailable agonists, was used as an example of a very challenging drug target. Hits are confirmed at the screening step due to the bell-shaped curve. This method is very similar to the quantitative HTS method (screening and hit confirmation at the same time), except that using this approach greatly decreases the data point number and can screen easily more than 100.000 biological relevant compounds.<ref>Helleboid S, Haug C, Lamottke K, et al. The Identification of Naturally Occurring Neoruscogenin as a Bioavailable, Potent, and High-Affinity Agonist of the Nuclear Receptor RORα (NR1F1). Journal of Biomolecular Screening. 2014;19(3):399-406. https://doi.org/10.1177/1087057113497095.</ref>

Switching from an orbital shaker, which required milling times of 24 hours and at least 10 mg of drug compound to a ResonantAcoustic mixer, Merck reported reduced processing time to less than 2 hours on only 1-2 mg of drug compound per well. Merck also indicated the acoustic milling approach allows for the preparation of high dose nanosuspension formulations that could not be obtained using conventional milling equipment.<ref name="a501">{{cite journal | last=Leung | first=Dennis H. | last2=Lamberto | first2=David J. | last3=Liu | first3=Lina | last4=Kwong | first4=Elizabeth | last5=Nelson | first5=Todd | last6=Rhodes | first6=Timothy | last7=Bak | first7=Annette | title=A new and improved method for the preparation of drug nanosuspension formulations using acoustic mixing technology | journal=International Journal of Pharmaceutics | publisher=Elsevier BV | volume=473 | issue=1-2 | year=2014 | doi=10.1016/j.ijpharm.2014.05.003 | pages=10–19}}</ref>

Whereby traditional HTS drug discovery uses purified proteins or intact cells, recent development of the technology is associated with the use of intact living organisms, like the nematode ''[[Caenorhabditis elegans]]'' and zebrafish ([[Zebrafish|''Danio rerio'']]).<ref>{{cite journal |vauthors=Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S, Heiss EH, Rollinger JM, Schuster D, Breuss JM, Bochkov V, Mihovilovic MD, Kopp B, Bauer R, Dirsch VM, Stuppner H | year = 2015 | title = Discovery and resupply of pharmacologically active plant-derived natural products: A review | doi = 10.1016/j.biotechadv.2015.08.001 | journal = Biotechnol. Adv. | volume = 33| issue = 8| pages = 1582–614| pmid = 26281720 | pmc=4748402}}</ref>

In 2016-2018 plate manufacturers began producing specialized chemistry to allow for mass production of ultra-low adherent cell repellent surfaces which facilitated the rapid development of HTS amenable assays to address cancer drug discovery in 3D tissues such as organoids and spheroids; a more physiologically relevant format.<ref name="pmid29743592">{{cite journal |last1=Kota |first1=S. |last2=Hou |first2=S. |last3=Guerrant |first3=W. |last4=Madoux |first4=F. |last5=Troutman |first5=S. |last6=Fernandez-Vega |first6=V. |last7=Alekseeva |first7=N. |last8=Madala |first8=N. |last9=Scampavia |first9=L. |last10=Kissil |first10=J. |last11=Spicer |first11=TP. |title=A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype. |journal=Oncogene |date=10 May 2018 |volume=37 |issue=32 |pages=4372–4384 |doi=10.1038/s41388-018-0257-5 |pmid=29743592|pmc=6138545 }}</ref><ref name="pmid29673279">{{cite journal |last1=Hou |first1=S. |last2=Tiriac |first2=H. |last3=Sridharan |first3=BP. |last4=Scampavia |first4=L. |last5=Madoux |first5=F. |last6=Seldin |first6=J. |last7=Souza |first7=GR. |last8=Watson |first8=D. |last9=Tuveson |first9=D. |last10=Spicer |first10=TP. |title=Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening |journal=SLAS Discovery |date=July 2018 |volume=23 |issue=6 |pages=574–584 |doi=10.1177/2472555218766842 |pmid=29673279|pmc=6013403 }}</ref><ref name="pmid28346088">{{cite journal |last1=Madoux |first1=F. |last2=Tanner |first2=A. |last3=Vessels |first3=M. |last4=Willetts |first4=L. |last5=Hou |first5=S. |last6=Scampavia |first6=L. |last7=Spicer |first7=TP. |title=A 1536-Well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids. |journal=SLAS Discovery |date=June 2017 |volume=22 |issue=5 |pages=516–524 |doi=10.1177/2472555216686308 |pmid=28346088|doi-access=free }}</ref>