High‐Throughput Screening (HTS) for theIdentification of Novel Antiviral Scaffolds
Manoj P. Jadhav
In recent past, the drug discovery field has experiencedmany challenges in the form of financial pressures and
decreased numbers of new drugs coming tomarket, which
has ultimately resulted in cost cutting and reducedexpenditure in research. There has been also a trend of
merging giant pharmaceutical companies with the aim of
short-term financial gains, which might affect scientificoutcomes in the long run. In an attempt to improve drug
discovery outcomes, various novel technologies have
been exploited; one of these which has gained muchattention is high-throughput screening (HTS).1 HTS is an
atomized technology for rapidly analyzing the activity of
hundreds and thousands of chemical compounds. Cou-pled with combinatorial chemistry and bioinformatics, it
has allowed potential hits to be quickly and efficiently
screened to find candidates that should be exploredfurther as possible new chemical entities.2 Since the
development of experimental antivirals through tradi-
tional drug discovery methods, there has been constantprogress in the discovery of antiviral drugs. Devastating
viral infections from human immunodeficiency virus
(HIV), hepatitis C virus (HCV), herpes simplex virus(HSV), hepatitis B virus (HBV), influenza virus, and
enveloped virus infections have been studied extensively
to find drugs. Currently, there are around 26 drugsapproved for treating HIV, and over 40 compounds are in
the advanced development stage for treating HCV.
However, there still remains an urgent need to findeffective treatments for viral infections like dengue, West
Nile, and yellow fever, among others. In the present
commentary, we try to shed some light on how thistechnology has contributed to the drug discovery
programs and how it can assist in finding antiviral drugs
in the twenty-first century.
How Does HTS Work?It is known that most drugs work by binding to a protein
target on or in a living cell in a biological system. As partof the drug discovery and development program, it is
critical to find molecules which will bind to selected
target proteins. For example, if one wishes to develop anantiviral drug which binds to and inactivates a particular
protein known to promote the diseases/infection, he need
to screen several compounds. There are few compounds
which weakly bind to the target protein, so these willserve as the starting point for generating a large number of
related compounds. Using knowledge of combinatorial
chemistry and bioinformatics, thousands of relatedcompounds can be quickly and automatically synthe-
sized. One can explore compounds from natural origin as
well. Those which bind the target best can be furtherevaluated, and may go on to pass the screens to be tested
in preclinical and clinical phases of drug development,
ultimately gaining approval as therapeutic candidates.Initial screening of these compounds for their binding
ability to the target protein is the job/function for HTS. A
critical step in HTS is to develop and validate an assay inwhich binding between a compound and a protein causes
some visible change/signal that can be automatically read
by a sensor.3,4 Generally, the change is observed in theform of emission of light by a fluorophore in a reaction
mixture. Typically, it can be done by attaching a
fluorophore to the target protein in such a way that itsability to fluoresce is diminished (quenched) when the
protein binds to another molecule. Another approach
could be to measure the difference in a particular propertyof light (polarization) emitted by bound versus unbound
fluorophores. Bound fluorophores are more highly
polarized, and this can be detected by sensors. Otherdetection methods are possible as well.
The details of HTS differ with various systems, but all
depend on automated or robotic systems to combine thechemicals and read the outputs. Microplates, (which are
plastic trays with multiple indentations, or wells), are
generally used to perform the reactions between targetprotein and the study compounds. Current HTS assays
can handle plates with 96, 384, 1,536, or even higher
numbers of wells at once, with extremely small volumesin each well (often 10mL or less). Small volumes have
many advantages, including minimizing the amount of
Clinical Pharmacologyin Drug Development3(2) 79–83
© 2014, The AmericanCollege of ClinicalPharmacologyDOI: 10.1002/cpdd.99
Division of Cardiovascular Medicine, College of Medicine, Universityof Florida, Gainesville, FL, USA
Submitted for publication 8 June 2012; accepted 9 December 2013
Corresponding Author:Manoj P. Jadhav, Division of Cardiovascular Medicine, College ofMedicine, University of Florida, Gainesville, FL 32610, USA(e‐mail: [email protected])
Guest Commentary
each compound used. This is especially important formany protein targets, which may be difficult and costly to
isolate and purify. In a single day, using fast robotic
systems combined with rapid reactions, one can screen10,000 or more compounds.5–7
What Are the Pitfalls in HTS?Different experts have expressed concerns about the HTS
technology. Some consider it to generate poor qualitydata, along with being expensive and time consuming,
and others even think it is an anti-intellectual and
irrational technique. There also exists another school ofthought which projects HTS to be a technology that
generates high quality data, which is better controlled
than data generated by lower throughput biological tests.HTS running laboratories generally implement robust
quality assurance methods such as “Z” trend monitoring,8
plate pattern recognition algorithms, liquid handler andreader performance monitoring, reagents specificity and
stability, DMSO compatibility, plate acceptance testing,
etc. Recent developments in low volume liquid handlingaccuracy, such as acoustic dispensing, detection instru-
mentation, and multimodal plate readers, have made the
data generation more accurate and reliable.9–11
HTS is also often criticized for its ability to test
compounds only at a single concentration during its
primary screen; however, current use of quantitative HTShas made it possible to test compounds at about seven
concentrations across four log-ranges of concentrations
(e.g., NIH chemical genomic center)12,13 With respect tothe anti-intellectual and irrational criticisms, it is widely
considered to be intellectually neutral; rather, it helps one
to perform the experiment more quickly. In actuality, thistechnique requires a broad knowledge base covering
various disciplines of biology, chemistry, engineering,
information technology and logistics, confirming itsintellectual credibility.
HTS is considered to produce less hits for many
targets, however, it has a success rate of more than 50% tofind hits.14 As compared to other hit discovery strategies
like fragment screening, structure based design, virtual
screening, etc. which have their limitations, so only by aclever critical integration of hit identification approaches
one can increase the chances of success in a developmen-
tal program.
Are There Any Success Stories?Yes, there are success stories from HTS. Although it is a
relatively young discipline of science, with the first
scientific paper in PubMed citing this technology in 1991,the technology grew slowly but steadily. As evident by
the slow drug discovery and development process, which
takes around 13.5 years to get a drug to market, it can be
said that only few HTS screens would have led to thedevelopment of the marketable drugs.15 In a recent study,
out of 58 drugs for which the starting lead was known,
which were approved by US Food and Drug Administra-tion (FDA) during 1991–2008 period, 19 of these had an
origin through HTS. Most of them belong to the
therapeutic category of anti-cancer, antiviral, anti-hypertension, etc. A classic example is maraviroc
(Selzentry), a chemokine receptor antagonist marketed
by Pfizer Ltd., which is the outcome of the strength ofHTS coupled with medicinal chemistry which optimized
the PK/PD properties of this candidate.16 This work
started with an HTS of around approx. 500,000compounds in 1997, and was completed in 2007 with
the US FDA approval for its indication inHIV. The screen
was performed using a chemokine receptor 5 radioligand-binding assay, which hit a weak agonist that had no
cellular antiviral activity, but gave a good starting point
for an extensive structural activity program. Subsequently,over 4,000 compounds were synthesized to eliminate
cytochrome P4502D6 (CYP2D6) and potassium voltage
gated channel (also known as HERG) undesired activityto get a potent, antiviral compound with slow offset
antagonist activity. The final product maraviroc demon-
strated safety and efficacy in clinical trials and receivedregulatory approval. This is a classical success story of the
medicinal chemistry efforts required in lead optimization
to turn an HTS hit into a therapeutic candidate.17
Examples of other drugs are provided in Table 1.
Status of HTS in AcademiaThe National Institute of Health (NIH) initiated NIH
RoadMap program in 2004 to advance chemical biologyby establishing the Molecular Library Initiative (MLI)
and Molecular Libraries Screening Center Network
(MLSCN). This has resulted in the set up of translational
Table 1. Drugs Approved With a Successful Use of HTSTechnology
DrugTherapeutic
Class Target ClassYear ofApproval
Gefitinib Anti‐cancer Tyrosine kinase 2003Erlotinib Anti‐cancer Tyrosine kinase 2004Tipranavir Anti‐HIV Protease 2005Sorafinib Anti‐cancer Tyrosine kinase 2005Sitagliptin Anti‐diabetic Protease 2006Lapatinib Anti‐cancer Tyrosine kinase 2007Ambrisentan Anti‐hypertensive GPCR 2007Maraviroc Anti‐HIV GPCR 2007Etravirine Anti‐HIV Reverse
transcriptase2008
Tolovaptan Hyponatraemia GPCR 2009
GPCR, G‐protein coupled receptor.
80 Clinical Pharmacology in Drug Development 3(2)
and chemical screening programs at various universitiesacross the country. Currently, there are at least 78
academic institutes that have HTS facilities to promote
chemical biology to produce useful hits, which can beused as starting points for finding solutions for treating
many diseases. Recently, MLSCN has been replaced
by Molecular Libraries Production Center Network(MLPCN), in order to combine HTS, structural activity
relationship (SAR) studies and probe development
efforts. All of these initiatives have been able to generatearound 360,000 compounds in various academic screen-
ing laboratories, with an ultimate aim to produce diverse
and high-quality chemical libraries. These facts indicate abright future for HTS screening in academic settings.18,19
What Is Happening in Anti‐Viral DrugDiscovery?In the following section, we would put light on some ofthe viruses which poses a larger public threat. Currently,
there are around 26 drugs approved for treating HIV, and
most of these drugs target one of the steps in the HIV-1replication cycle, that is, HIV-1 fusions, reverse
transcriptase, or proteases. However, viral variants
resistant to one drug of a particular class often exhibitsome level of cross-resistance to other drugs within the
same class. Therefore, therapeutic options are often
limited in already-treated patients, leading to cross-resistance. Keeping these problems in mind, a proof-of-
concept regarding clinical efficacy has been demonstrated
for three new targets in the HIV-1 replication cycle,
that is, HIV-1 co-receptors, HIV-1 gp120 and HIV-1integrase. Current efforts are also focused on a once-a-day
combination pill consisting of a combination of drugs
with unique mechanisms of action and distinct resistanceprofiles.20 Some of the details on viruses, model systems
used, and targets for antiviral scaffold identification are
summarized in Table 2.Over 50 compounds are in the advanced development
stage for treating HCV. Viral polymerases and proteases
are targets of choice, and have been validated by usinginhibitors of HIV reverse transcriptase, protease, hepatitis
B polymerase, and herpes virus polymerase as antiviral
drugs. Recently telaprevir (Vertex Pharmaceuticals) andboceprevir (Merck Ltd.) were approved for treatment of
HCV infection. Two other first generation protease
inhibitors namely TMC435 (Tibotec Pharmaceuticals)and BI201335 (Boehringer Ingelmeim) are in phase III of
development. Both are taken once a day, have fewer side
effects and might be even more potent than boceprevirand telaprevir. Further, the second-generation protease
inhibitor MK-5172 is expected to be devoid of cross-
resistance issues with other drugs for this class and mightbe effective across multiple genotypes. There are other
phase III studies underway for the treatment of HCV.21
HCV belongs to the Hepacivirus genus within theFlaviviridae family. Another virus of concern from the
same family is dengue virus. So, it would be worthwhile
to use the knowledge generated and the strategies gainedfrom the successful drug development process for
HCV22–24 for the discovery of antiviral agent for dengue.
Dengue fever is the most frequent arthropod-borne viral
Table 2. Details of Viruses, Model Systems Used, and Target for Antiviral Scaffold Identification
Virus Model System Used Target Refs.
Dengue virus Cytopathic effect‐based assay DENV NS2B/NS3 protease Yang et al30
Immunofluorescence stainingof DENV glycoprotein
Transporters, receptors, and protein kinases Shum et al31
Fluorescence‐based alkalinephosphatase‐coupledpolymerase assay
RNA‐dependent RNA polymerase Niyomrattanakit et al26
HIV Fluorescence‐based assay Inhibitors of HIV reverse‐transcriptaseDNA polymerase
Cauchon et al32
Fluorescence resonanceenergy transfer (FRET)
HIV—gp41 Xu et al33
Dual reporter assay Cytotoxicity Westby et al34
West Nile virus Cell viability assay‐(CellTiter‐Glo1) Signal is proportional to the quantity ofATP in host cells
Chung et al35
Luciferase‐expressing replicon,virus‐like particles, andfull‐length virus assay
Viral entry, replication, and virion assembly Puig‐Basagoiti et al36
Hepatitis C Enzyme assay Selective inhibitors of HCV NS5B polymerase Ontoria et al37
Cell‐based HCV infection assay HCV NS3 peptide cleavage sequence Yu et al38
Reporter replicon system HCV RNA replication inhibitors Hao and Duggal23
Cell‐based assay Inhibitors against HCV genotypes 1a and 1b Mondal et al39
Jadhav 81
disease of humans, with almost half of the world’spopulation at risk. Its high prevalence, lack of effective
vaccine, and absence of specific treatment strongly
suggests it be treated as a global public health concern.Among the potential targets to find the drugs for dengue is
through inhibition of viral entry, fusion of viral
membrane with the host and its replication, etc. Otherpotential viral targets that have gained attention are non-
structural proteins 3 and 5 (NS3, NS5), which have
important roles in genome replication, also it isworthwhile to focus on their protease domains as to
find protease inhibitors.
Various strategies have been exploited to find drugsfor dengue, including luciferase reporter assay, fluores-
cence-based alkaline phosphatase assay, high content
cell-based assay, renilla luciferase reporter replicon, andcytopathic effect-based assay.25–29 There are ample
studies reported wherein HTS screens have been used
to identify hits for dengue. The Novartis Institute ofTropical Diseases at Singapore has made significant
progress in this direction, and it is expected that they will
come up with a promising agent soon. The structuralsimilarities between different flaviviruses (e.g., West
Nile, Yellow fever, etc.), allow the knowledge generated
and strategies applied in one disease to be exploited tofind possible hits for the other diseases. This warrants a
coordinated interdisciplinary effort to find solutions. In
this endeavor, HTS can play a vital role in screeningcompounds both from synthetic as well as natural origins.
In summary, HTS has gained recognition as a mature
scientific discipline in pharmaceutical industry researchpost human genome era. It has hastened speed, capacity,
and outcome in the drug development process. The
advancements in HTS technology in the form offluorescent cell barcoding, quantitative HTS coupled
with different type of assay approaches like luciferase-
expressing replicon, virus-like particles, and full-lengthvirus assay have tremendously helped the drug discovery
process especially in area of antiviral research. In future,
this technology may provide novel tools for basicvirology research as well as potential new therapeutics
for a variety of viral infections affecting millions of
people worldwide.
Declaration of Conflicting Interests
The author declares that there is no conflicts of interest and no
finances received from any source.
References
1. Ionov Y. A high throughput method for identifying
personalized tumor-associated antigens. Oncotarget.
2010;1:148–155.
2. Mayr LM. Bojanic D. Novel trends in high-throughput
screening. Curr Opin Pharmacol. 2009;9:580–588.
3. Eggeling C, Brand L, Ullmann D, Jager S. Highly sensitive
fluorescence detection technology currently available for
HTS. Drug Discov Today. 2003;8:632–641.
4. Pope AJ, Haupts UM, Moore KJ. Homogeneous
fluorescence readouts for miniaturized high-throughput
screening: theory and practice. Drug Discov Today. 1999
4:350–362.
5. Fox JT, Myung K. Cell-based high-throughput screens for
the discovery of chemotherapeutic agents. Oncotarget.
2012;3:581–585.
6. Clemons PA, Tolliday NJ, Wagner BK, eds. BT Cell-based
assays for high-throughput screening. Methods and proto-
cols. Series: Methods in molecular biology, Vol. 486. New
York, USA: Humana Press, a part of Springer Science
Business Media, LLC; 2009.
7. Macarron R, Hertzberg RP. Design and implementation of
high throughput screening assays.Mol Biotechnol. 2011;47:
270–285.
8. Zhang JH, Chung TD, Oldenburg KR. A simple statistical
parameter for use in evaluation and validation of high
throughput screening assays. J Biomol Screen. 1999 4:
67–73.
9. Makarenkov V, Zentilli P, Kevorkov D, et al. An efficient
method for the detection and elimination of systematic
error in high-throughput screening. Bioinformatics. 2007;
23:1648–1657.
10. Coma I, Clark L, Diez E, et al. Process validation and screen
reproducibility in high-throughput screening. J Biomol
Screen. 2009;14:66–76.
11. Taylor PB, Ashman S, Baddeley SM, et al. A standard
operation procedure for assessing liquid handler perfor-
mance in high-throughput screening. J Biomol Screen.
2002;7:554–569.
12. Michael S, Auld D, Klumpp C, et al. A robotic platform for
quantitative high-throughput screening. Assay Drug Dev
Technol. 2008;6:637–657.
13. Entzeroth M, Flotow H, Condron P. Overview of high-
throughput screening. Curr Protoc Pharmacol. 2009;
Chapter 9:Unit 9.4. doi: 10.1002/0471141755.ph0904s44
14. Fox S, Farr-Jones S, Sopchak L, et al. High-throughput
screening: update on practices and success. J Biomol
Screen. 2006;11:864–869.
15. Paul SM, Mytelka DS, Dunwiddie CT, et al. How to
improve R&D productivity: the pharmaceutical industry’s
grand challenge. Nat Rev Drug Discov. 2010;9:203–214.
16. Dorr P,WestbyM, Dobbs S, et al. Maraviroc (UK 427,857),
a potent, orally bioavailable, and selective small-molecule
inhibitor of chemokine receptor CCR5with broad-spectrum
anti-human immunodeficiency virus type 1 activity.
Antimicrob Agents Chemother. 2005;49:4721–4732.
17. Macarron R, Banks MN, Bojanic D, et al. Impact of high-
throughput screening in biomedical research. Nat Rev Drug
Discov. 2011;10:188–195.
18. Silber BM. Driving drug discovery: the fundamental role of
academic labs. Sci Transl Med. 2010;2:30cm16.
82 Clinical Pharmacology in Drug Development 3(2)
19. Frye SV. The art of the chemical probe. Nat Chem Biol.
2010;6:159–161.
20. Westby M, Nakayamab GR, Butler SL. Cell-based and
biochemical screening approaches for the discovery of
novel HIV-1 inhibitors. Antiviral Res. 2005;67:121–140.
21. Schlutter J. Therapeutics: new drugs hit the target. Nature.
2011;474:S5–S7.
22. Yu X, Sainz B Jr, Uprichard SL, et al. Development of a
cell-based hepatitis C virus infection fluorescent resonance
energy transfer assay for high-throughput antiviral com-
pound screening. Antimicrob Agents Chemother. 2009;53:
4311–4319.
23. Hao W, Duggal R. High-throughput screening of HCV
RNA replication inhibitors by means of a reporter replicon
system. Methods Mol Biol. 2009;510:243–250.
24. Schreiber SL, Nicolaou KC, Davies K. Diversity-oriented
organic synthesis and proteomics. New frontiers for
chemistry & biology. Chem Biol. 2002;9:1–2.
25. Xie X, Wang QY, Xu HY, et al. Inhibition of Dengue virus
by targeting viral NS4B protein. J Virol. 2011;85:11183–
11195.
26. Niyomrattanakit P, Abas SN, LimCC, et al. A fluorescence-
based alkaline phosphatase-coupled polymerase assay for
identification of inhibitors of dengue virus RNA-dependent
RNA polymerase. J Biomol Screen. 2011;16:201–210.
27. Shum D, Smith JL, Hirsch AJ, et al. High-content assay to
identify inhibitors of dengue virus infection. Assay Drug
Dev Technol. 2010;8:553–570.
28. Qing M, Liu W, Yuan Z, Gu F, Shi PY. A high-throughput
assay using dengue-1 virus-like particles for drug discovery.
Antiviral Res. 2010;86:163–171.
29. Che P, Wang L, Li Q The development, optimization and
validation of an assay for high throughput antiviral drug
screening against dengue virus. Int J Clin Exp Med. 2009;
2:363–373.
30. Yang CC, Hsieh YC, Lee SJ, et al. Novel dengue virus-
specific NS2B/NS3 protease inhibitor, BP2109, discovered
by a high-throughput screening assay. Antimicrob Agents
Chemother. 2011;55:229–238.
31. Shum D, Smith JL, Hirsch AJ. High-content assay to
identify inhibitors of dengue virus infection. Assay Drug
Dev Technol. 2010;8:553–570.
32. Cauchon E, Falgueyret JP, Auger A, et al. A high-
throughput continuous assay for screening and characteri-
zation of inhibitors of HIV reverse-transcriptase DNA
polymerase activity. J Biomol Screen. 2011;16:518–524.
33. Xu Y, Hixon MS, Dawson PE, et al. Development of a
FRET assay for monitoring of HIV gp41 core disruption.
J Org Chem. 2007;72:6700–6707.
34. Westby M, Nakayama GR, Butler SL, et al. Cell-based and
biochemical screening approaches for the discovery of
novel HIV-1 inhibitors. Antiviral Res. 2005;67:121–140.
35. Chung DH, Ardecky R, Ganji SR, et al. A cell based assay
for the identification of lead compounds with anti-viral
activity against West Nile virus. 2010 Feb 27 [Updated
2010 Oct 4]. In: Probe reports from the NIH Molecular
Libraries Program [Internet]. Bethesda (MD): National
Center for Biotechnology Information (US); 2010. Available
from: http://www.ncbi.nlm.nih.gov/books/NBK50698/ (Ac-
cessed November 15, 2012).
36. Puig-Basagoiti F, Deas TS, Ren P, et al. High-throughput
assays using a luciferase-expressing replicon, virus-like
particles, and full-length virus for West Nile virus drug
discovery. Antimicrob Agents Chemother. 2005;49:4980–
4988.
37. Ontoria JM, Rydberg EH, Di Marco S, et al. Identification
and biological evaluation of a series of 1H-benzo[de]
isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B
polymerase inhibitors. J Med Chem. 2009;52:5217–5227.
38. Yu X, Sainz B Jr, Uprichard SL. Development of a cell-
based hepatitis C virus infection fluorescent resonance
energy transfer assay for high-throughput antiviral com-
pound screening. Antimicrob Agents Chemother. 2009;53:
4311–4919.
39. Mondal R, Koev G, Pilot-Matias T, et al. Development of a
cell-based assay for high-throughput screening of inhibitors
against HCV genotypes 1a and 1b in a single well. Antiviral
Res. 2009;82:82.
Jadhav 83