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: manojpjadhav@yahoo.com)

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