**Abstract**

The process of drug discovery is challenging and a costly affair. It takes about 12 to 15 years and costs over \$1 billion dollars to develop a new drug and introduce the finished product in the market. With the increase in diseases, virus spread, and patients, it has become essential to invent new medicines. Consequently, today researchers are becoming interested in inventing new medicines faster by adopting higher throughput screening methods. One avenue of approach to discovering drugs faster is the High-Throughput Screening (HTS) method, which has gained a lot of attention in the previous few years. Today, High-Throughput Screening (HTS) has become a standard method for discovering drugs in various pharmaceutical industries. This review focuses on the advancement of technologies in High-Throughput Screening (HTS) methods, namely fluorescence resonance energy transfer (FRET), biochemical assay, fluorescence polarization (FP), homogeneous time resolved fluorescence (HTRF), Fluorescence correlation spectroscopy (FCS), Fluorescence intensity distribution analysis (FIDA), Nuclear magnetic resonance (NMR), and research advances in three major technology areas including miniaturization, automation and robotics, and artificial intelligence, which promises to help speed up the discovery of medicines and its development process.

**Keywords:** Drug discovery and development, High-Throughput Screening, fluorescence resonance energy transfer, biochemical assay, fluorescence polarization (FP), homogeneous time resolved fluorescence (HTRF), Fluorescence correlation spectroscopy (FCS), Fluorescence intensity distribution analysis (FIDA), Nuclear magnetic resonance (NMR), miniaturization, automation and robotics, and artificial intelligence

### **1. Introduction**

It takes about 12 to 15 years and costs over \$1 billion dollars to develop a new drug and introduce the finished product in the market. Moreover, the process is highly complex and costly as huge investments are made into technology [1, 2]. There is a need to reduce costs, increase efficiency and introduce drugs conveniently and faster to the market by higher throughput methods. The High-throughput screening method screens millions of chemical and biological compounds in a short interval of time. It is an automated process and screens many biological or chemical compounds for their therapeutic potential. The

High-throughput screening method efficiently accelerates the discovery of drugs, which are of potentially great therapeutic promise compared with other screening methods [3–5].

This review highlights the types of High-throughput screening assays and different detection techniques such as fluorescence resonance energy transfer (FRET), biochemical assay, fluorescence polarization (FP), homogeneous time resolved fluorescence (HTRF), Fluorescence correlation spectroscopy (FCS), Fluorescence intensity distribution analysis (FIDA) [6], Nuclear magnetic resonance (NMR), and research advances in three major technology areas including miniaturization, automation and robotics, and artificial intelligence, which has shown great promise to speed the discovery of medicines and its development process [7, 8].

#### **1.1 Understanding high-throughput screening process for the discovery of drugs**

In the high-throughput screening process, many compounds are screened to find potential candidate compounds that efficiently affect a biological target. These so-called candidate compounds are referred to as 'hits.' As an example, the high-throughput screening process successfully identified a potent pan-SRC kinase inhibitor now known as 'Dasatinib, BMS-354825' for the biological target of diabetes. The high-throughput screening processes involve various detection methods such as robotics or plate readers and corresponding software to process and analyze the data obtained. Once the 'hits' are discovered, further analysis in the high-throughput screening process can enable one to understand the potential optimization of the 'hits' achieved during the first screening round [9–11].

It is essential to understand that the high-throughput screening method alone cannot wholly evaluate a potential drug as toxicity studies are further needed for this evaluation. Instead, high-throughput screening is essential to eliminate the time that would have been wasted on investigating compounds that had little or no desired effect on the biological target. Through the utilization of this automated screening process, millions of compounds can together be screened and compounds with no or poor effects eliminated [10].

Basically, in the process of high-throughput screening HTS high number of effectors and biological modulators are screened and assayed against specific and selected targets. The high-throughput screening HTS process ensures that the time taken to screen large compound libraries is reduced and the whole drug discovery process is speeded up. In this manner, the high-throughput screening HTS method can be capable of screening more than thousand compounds per day [12]. It is ideally a mechanism-based approach where compounds are screened to provide improved drugs. High-throughput screening process precisely focuses on single mechanism contributing to identification of target specific compounds. The high-throughput screening HTS assays helps to screen various types of libraries such as genomics, protein, combinatorial chemistry, and peptide libraries [10]. The high-throughput screening HTS and assay method includes various steps such as preparation of reagents, target identification, compound management, assay development, and high throughput library screening, which are performed with extreme care and precision. The detail steps are as follows [13].

Firstly, targets are selected. There are presently around 500 targets that are being utilized by various companies. Among these targets, cell membranes receptors, mostly G-protein coupled receptors are commonly used and comprise the largest group with 45% of the total, followed by Enzymes (28%), hormones (11%), unknowns (7%), ion-channels (5%), nuclear receptors (2%), and DNA (2%). Off late pharmaceutical companies are interested to analyze compounds that interfere or modulate the function of GPCRs [13].

*Design and Implementation of High Throughput Screening Assays for Drug Discoveries DOI: http://dx.doi.org/10.5772/intechopen.98733*

Undoubtedly, the integration of different compound libraries with wide chemical diversity in the high throughput screening method is a potential solution for massive drug discovery. The identification of good hits via the high throughput screening method can effectively reduce the time frame of discovering drugs in the process. However, for the screening technique to be successful various factors are dependent on it. Various factors that are essential to be considered are quality and number of validated targets, diversity and number of compounds, and the capability to screen compounds in a cost effective and timely manner using robust informative assays. Not just that, there are certain limitations of the high throughput screening method that defines the pharmacological properties of active compounds such as, synthetic chemistry for lead optimization and the low throughput of secondary assays. This major drawback causes a major hindrance to the overall identification rate of potential candidates for clinical evaluation. Due to this reason, researchers and scientists are trying to develop better technological solutions to overcome these challenges.

#### **1.2 Application of high throughput screening (HTS) in drug discoveries**

Applications of High-throughput screening (HTS) method in drug discoveries are detailed below

#### **1.3 In screening**


Generally, High-throughput screening process is carried pout via a microliter plate. Today, modern micro plates for HTS assays are performed in automationfriendly microliter plates with a 96, 384, 1536 or 3456 well format. The so called wells successfully comprise of experimentally useful matter, often an aqueous solution of dimethyl sulfoxide (DMSO) and some other chemical compound, the latter of which is different in each well across the plate [14].

Today, in most of the drug discovery labs, the collection of libraries has increased from 400,000 to more than 1 million compounds. In order to screen these high numbers of compound libraries automated 384 wells or higher density single compound test formats are used. Ideally, the primary screen is responsible and designed for rapid identification of hits from this library of compounds. The aim is to achieve a minimum number of false positives and maximum number of confirmed hits. Not to mention, the hit rates generally range between 0.1 – 5%, depending on the assay. The hit range or number also depends on the cutoff parameters that are set by the researchers, or the dynamic range of a given assay. These Primary screens run in multiples of single compound concentrations. The results of the primary screening method are expressed in terms of percent activity as a negative (0 percent) and a positive (100 percent) control. The achieved Hits are further retested, generally independently from the first assay. After retesting, if a compound displays the same activities, it is accepted as a confirmed hit, and the process go through secondary screening or lead optimization. The results obtained from the secondary screening method are used to decide and further filter the substances that will make it on to clinical trials [13].

The combination of screening methods with bioinformatics, allows potential drugs to be efficiently and quickly screened, and hence discovering drugs at a faster speed and, which can be explored in more detail. The Initial screening of these compounds for their binding ability is the main role of high-throughput screening method. The high-throughput screening process generally involves developing tests, or assays, where in the potential compounds are made to bind with proteins, causing visible change that can be automatically read by a sensor. Generally, this change is achieved by light emissions by a fluorophore in the reaction mixture. The way the process works out is that, fluorophores are attached to target proteins in such a way that its ability to fluoresce is diminished (quenched) when the protein binds to another molecule. Then a different system measures the difference in polarization, which is a property of light, emitted by unbound versus bound fluorophores. Usually, bound fluorophores are highly polarized and hence can be easily detected by sensors. Various detection technologies for high throughput s screening are available today these includes time-resolved fluorescence (TR-FRET), fluorescence resonance energy transfer (FRET), fluorescence polarization, luminescence and absorbance. Not to mention these methods required efficient, highly sensitive, and versatile multi-mode micro plate readers [13].

Generally, libraries are referred as sets of compounds produced by combinatorial chemistry. Depending on how the solid-phase are handled, these compounds may be either mixtures or individual compounds. In biological assays range of compounds present in the libraries are tested as follows.


#### **1.4 Test of mixture in solutions**

In this test method the compounds are cleaved from the beads and tested in solution. Sometimes, it is a tedious task to find the compounds that are active by observing the pharmacological screen. In order to carry out successful identification of the most active compounds, it is essential to resynthesize the componenets individually. In this manner the process of screening and resynthesizing in an iterative manner is one of the most successful and simple methods for the identification of most potent components from libraries.

#### **1.5 Test of individual compounds in solutions**

A second method of testing compounds is the separation of the beads manually into individual wells and cleaving the compounds from the solid-phase. These compounds are then tested as individual entities.

*Design and Implementation of High Throughput Screening Assays for Drug Discoveries DOI: http://dx.doi.org/10.5772/intechopen.98733*


#### **Table 1.**

*Based on 96 well format.*

#### **1.6 Test compounds on the beads**

Another method for screening is testing on the beads. This is carried out by the application of fluorescent assay or colorimetric technique. In this process appropriate beads can be chosen by fluorescence or color, and picked out by micromanipulation. Further the the product structure of the active compounds, if a peptide, can be determined by sequencing on the bead. Whereas, non-peptide structures can be identified by one of the tagging methods.

#### **1.7 Applications**

High-throughput technology finds wide applications in areas other than drug development. These include the following


#### **1.8 Traditional screening vs. high-throughput**

Traditional methods were time consuming as compared to high-throughput discovery. The table below demonstrates the differences between traditional methods and high-throughput methods. As shown in **Table 1**, screening ability of high-throughput screening ability increased 50 times on the low end, and 200 times on the high end as compared to traditional screening [3]. Leading to an increase of efficacy and accuracy, in High-throughput screening method. Moreover in highthroughput screening method minimal amounts of test compounds are used [12].

#### **2. Types of high throughput assays**

Assays are segregated into cell-based assays and biochemical assays. Biochemical assays are further segregated into homogeneous and heterogeneous assays [15].

#### **2.1 Homogeneous assay**

Homogeneous assay measurement is a single step process, where reagents are added in a single stage or multiple steps. Steps involved are fluid addition, incubation and readings. The homogeneous assay measurement is characterized by the interaction between the surrounding environment and the analyte. The homogeneous assay measurement method can be coupled with different detection techniques such as fluorescence, radiometric etc. for HTS. The advantageous feature of the homogeneous assay measurement method is that it is simple and involves minimum steps thereby contributing to reduce cost and minimum robotic complexity required in automation. One of the drawback of the homogeneous assay measurement method is that there are interference in measurements as it is carried out in the presence of other assay components and having a signal to background ratio of less than 10 [15].

#### **2.2 Heterogeneous assays**

Heterogeneous assays measurement method is a bit more complicated than the homogeneous assay method as it involves a few additional steps such as filtration, centrifugation etc. These additional steps ensures that the component(s) to be measured are separated from rest of the components, which may cause interference in assays measurement method and hence contributing to high signal to background ratio. A thumb rule always followed is that when homogeneous assay fails or high signal to background ratio is required, heterogeneous assay measurement method is generally carried out [15].

#### **2.3 Biochemical assays**

Biochemical assays are protein, enzyme-based, or receptor assays that utilizes a designated target in a more purified form. Generally speaking, biochemical assays are frequently carried out using scintillation proximity assay (SPA), radiometric, colorimetric fluorescence detection techniques. In the Scintillation Proximity Assay technology binding reactions are assayed without carrying out the filtration or washing process step. In this technique radioactive labels emitting electrons at about 10 μm in water are used to carry out the assay. Generally, SPA technique is a preferred method for all surface cell receptors when high binding and low receptor density is required [15].

One of the biochemical assays technique is Fluorescence resonance energy transfer. This technique is further summarized in the next sections [15].

#### **2.4 Fluorescence resonance energy transfer (FRET)**

FRET technique is a non-radiative quantum mechanical process, where energy is transferred from an excited donor fluorophore to a suitable acceptor fluorophore. Here, energy from incident light is absorbed onto the donor fluorophore and it is transferred to nearby acceptor molecule [15].

There are certain conditions that must be fulfilled for an effective FRET assay:


*Design and Implementation of High Throughput Screening Assays for Drug Discoveries DOI: http://dx.doi.org/10.5772/intechopen.98733*


#### **2.5 Fluorescence polarization (FP)**

The Fluorescence polarization technique is widely used in high throughput screening method. In this technique when light is irradiated to the fluorophore, it gets excited and emits light in same polarized plane. The fluorophore remains steady throughout the excitation state. However, if the fluorophore changes its position i.e., it rotates during excitation state, it emits light in different plane (depolarized). Larger molecule tends to show little movement while smaller molecule rotates quickly and giving high and low polarization value [15, 16].

#### **2.6 Applications**


#### **2.7 Homogeneous time resolved fluorescence (HTRF)**

The HTRF method is a combination of time resolved measurement (TR) of fluorescence and standard FRET technology, thereby allowing the elimination of shortlived background Fluorescence, which occurs due to interfering materials in the sample and allowing a delay of approximately 50 to 150μseconds between the initial excitation and fluorescence measurement. Homogeneous time resolved fluorescence method utilizes europium cry ptate (Eu3 + cryptate) as fluorescent energy donor, which are rare earth complexes consisting of a macrocycle within which a Eu3+ ion is tightly embedded. This cage behaves as an antenna, collecting and transferring energies to the Eu3+ ion that eventually releases this energy with a specific long lived fluorescent pattern. Cryptate comprise of cross-linked allophycocyanin or XL665, a phycobilli protein pigment purified from red algae as acceptor [15].

In a recent research study HTRF was used as a screening application for the assay of tyrosine kinase and screening against tumor necrosis factor receptor in a 384 well microplate.. It was observed that (HTRF) was similar and related to fluorescence intensity techniques. The detector was gated for a short time period of

(10 ns) - > and the initial burst of fluorescence was not measured. Post this gating period the longer lasting fluorescence of the sample was measured. HTRF is effectively used to enhance sensitivity levels [15].

Applications of HTRF and FRET methods:

