Miniaturization In Rational drug design of Pharmaceuticals – A Review

Introduction

In early phases of development, only small amounts of the active pharmaceutical ingredient (API) are available. With difficult drugs however, many different formulations are necessary to achieve adequate bioavailability. Therefore, low API consumption for each formulation creates a substantial developmental advantage. This can be achieved by miniaturized equipment for the most relevant pharmaceutical technologies. Until a few years ago, the commercially available equipment required minimal batch sizes of 50 – 100 g for nearly all formulation technologies. Number of small scale equipment increased considerably in the meantime, but in most cases, the basic technologies are still designed for large scales, only the formulation part is smaller. Therefore, this equipment is still very expensive, heavy, and requires a lot of lab space and cannot be located easily into containments which is necessary for

toxic or highly active APIs. Therefore, miniaturized equipment for the most relevant formulation technologies of oral dosage forms was developed at the group new technologies of Boehringer Ingelheim (BI).

The need to discover new pharmaceuticals Rapidly and at a lower cost continues to change The way drug discovery is practiced within the Pharmaceutical industries. Increasingly, drug developers are confronting the need to streamline their processes, improve the robustness of their screening operations and enhance the quality of early development candidates. HTS of large collections of compounds against therapeutic targets is increasingly employed as part of an early-stage strategy for  identifying active chemo types, which can eventually be developed into marketable drugs. The number of compounds screened in HTS laboratories runs into the millions3. As the number of therapeutic targets increase, so will demand for HTS, creating pressure to improve efficiency. Ultra-HTS (huts), in which more compounds are screened at lower cost and in less time, has been a major goal. The expansion of the role of uHTS in the drug development process creates the need for new technologies. Technology that enables the miniaturization of screening assays has been one route towards accomplishing µHTS. Miniaturized assays consume less reagents and compounds, and reduce the cost of screening. Miniaturized assays can also be performed faster and therefore reduce the time required to complete primary screens.

Assay miniaturization has followed an evolutionary process, starting with the movement of tests away from milliliter volumes in test tubes, and towards micro liter volumes  in the  standard 96- well micro-plate format¹. This evolution has continued with the increased utilization of 384-well plates, which enables assays to be performed in the range of 10–20µl. The next logical step is the development of assays in the submicroliter volume range. Assays performed in 1536-well plates at volumes of  ‹ 2 µl would significantly reduce the cost and time of screening. The greatest impact would be for screens performed with very large compound collections². Specific challenges to the implementation of a miniaturized screening platform based on the use of 1536-well plates include the development of instruments for detection and fluid handling.

Miniaturized assays performed at volumes of 2 µl or less require fluid handling equipment capable of operating reliably in the submicroliter volume range. Assay plates also need to accommodate the volumes. Many of these challenges are being actively pursued by the suppliers of screening instruments and commodities. Detectors capable of reading 1536-well plates have been on the market for  more than an year  now . Although dispensers capable of working in the submicroliter volume range are available now, fluid handling remains the greatest barrier to  implementation of miniaturized uHTS³.

This article describes the problems and challenges associated with fluid handling for miniaturized µHTS. The focus will be on approaches that have been undertaken to develop instruments for fluid handling in the submicroliter volume range. We will also discuss the impact of plate design for assay miniaturization in regard to fluid handling at these volumes.

Current Status of Knowledge

As HTS progresses into the next century, a concomitant consolidation of the complex synergy between associated developments in chemistry, biology, engineering and informatics for  lead discovery is necessary. It is anticipated that, in addition to providing innovative solutions to technological challenges, this consolidation must also enable incorporation of  µHTS technologies into the infrastructure of the pharmaceutical industry. It has been estimated that industrial screening demands will require the number of new Chemical entities introduced per year are to be tripled, necessitating a threefold increase in the  speed of current screening technology.

Miniaturization provides as a key sourse of  keeping and maintaining the constant pace with genomics because it enables proportionately more targets and samples to be screened per unit time. Because of the small amounts  of  compounds and  reagents used  in the  miniaturized system, this increase in screening speed can be achieved without an increase in associated R&D costs⁴. As a result of the benefits mentioned  previously, miniaturization setss a key foundation of screening philosophy.

Combining miniaturized technology with developments in automation, sensitive signal detection,  plate formats, automated compound-delivery and data management results in high efficiency, and cost-effective, integrated miniaturized  µHTS systems. This review will concentrate on the  recent major developments that have occurred in the µHTS arena over the past few years. Adaption of assay designs for µHTS running conditions will also be discussed.

Assay miniaturization

Assays present a significant demand On  µHTS systems. Established biochemical Assays  should be readily adaptable to miniaturized formats to shorten overall screening cycle-time. Lab-on-a-chip and micro scale total analysis systems are highly miniaturized⁵. Such systems promise assay volumes at the peculator level and throughputs that will easily exceed 100,000 assays per day. In addition to increasing assay throughput by incorporating low volume, high density formats, further  improvements can be achieved through the use of multiplexing strategies.

Multiplexing involves the detection of multiple screening-parameters simultaneously or in rapid sequence; these parameters might include the fluorescence polarization, intensity, lifetime and emission wavelength of a single or multiple species⁶. In many target classes, it is now possible to design assay systems that involve mixing the components, incubating to a suitable end-point or equilibrium and measuring a detection signal. This homogeneous ‘mix and measure’ type of assay is ideal for HTS. Functional cellular assays in miniaturized format are increasing in importance as primary screening assays⁷. Although cell-based assays using reporter genes have proved effective as a µHTS format, detecting more immediate responses to target-protein activation provides several advantages, including shorter assay duration and fewer false-positives from non-specific interactions. Recent advances in miniaturization technology and molecular biology have made it possible to monitor, for example, the presence of second messengers (Ca2+, camp, instill triphosphate), phosphorylation of intermediate signaling molecules or sub cellular translocation⁸.

Assay miniaturization is the process of establishing optimal assay conditions for the microliter volume range that is necessary for the screening of high-density-well plates, thus minimizing reagent consumption and reducing

storage capacity (Table 1). Several issues need to be tackled during assay miniaturization:

• Appropriate and accurate liquid handling (e.g. dispensing of cells through narrow-bore pipettes presents particular difficulties);
• Minimizing evaporation effects;
• Ensuring comparable assay sensitivity (dynamic range, binding constant K_id, IC₅₀) and screening statistics;
• Tackling the increased surface : volume ratio, which increases adsorption effects;
• Reproducing the conditions to be encountered on the HTS system as closely as possible (i.e. tackling issues such as reagent stability, kinetics of enzyme reactions. and sedimentation and viability of cells); and Using full high-density-plate layouts in assay development and assay miniaturization⁹.

Solutions to overcome these problems at the nanoscale level include altering the concentration and/or the order of addition of assay reagents (e.g. by adding ‘sticky’ reagents last) and adding detergents that reduce non-specific binding in a typical concentration range between 0.01% and 0.5%.

Automated assay optimization (AAO), which takes advantage from the statistical design of experiments (DOE), is a key method in the reduction of assay parameters and is ideal for application to high-density-plate formats. If used properly, AAO enables uHTS laboratories to reduce assay optimization timelines and to optimize ‘throw away’ assays that would not be a subject of a screening run under usual conditions.

The translation of assay protocols from assay development via assay miniaturization to the µHTS platform is a challenge that must not be underestimated. The use of bench top workstations with hardware components identical to those installed within the µHTS system is the key factor for running a huts factory

successfully¹⁰.

Table1:AdvantagesanddisadvantagesofminiaturizeduHTS

M miniaturized uHTS Key Features	Positive	Negative
Compound logistics	Saving of precious compounds	Reformatting step from 96, 384 to ≥1536-well plates necessary
	High-density storage possible
	Potential for automated fractionation of natural
	Extracts into screening plates
Assay miniaturization	Saving precious target or legend	Necessary effort because of altered surface: volume
		ratio and evaporation issues(adsorption effects) in
		high-density formats
		Solution: Bench top workstations for assay
		development, consisting of the same liquid-handling
		hardware as screening devices necessary for assay
		compatibility tests
Plate formats	High compound density (less process steps,	Increased demand on plate manufacturing
	more parallelization) allows enhanced	Precise plate adjustment in dispensing and reading
	throughput 1536-well plates already	System necessary
	compatible with many systems
	Plates of 2080 and 3456 wells already
	routinely applied
Liquid handling	Precise and reliable nanoliter handling with	Cell handling difficult, but possible
	piezoelectric drop-on-demand systems
	96 or 384-Well parallel dispensing enhances
	velocity of assay assembly
Detection systems	Confocal detection independent	Restricted to fluorescence or luminescence based
	of miniaturization. Imaging technologies	readouts
	applicable to fast 1536-well reading	M macroscopic fluorescence sensitive to
	Fast 96 or 384-well parallel reading	miniaturization
Automation	Fully automated screening platforms	Sealed humid atmosphere as protection against
	(integrated or modular) available for screening	evaporation necessary
	in 1536, 2080 or 3456-well plates
Data management	Automation of data handling is	Increased efforts in database administration and data
	worthwhile because of high data amounts	mining

Acknowledgement

Sultan-Ul-Uloom College Of Pharmacy, Banjara Hills, Road No.3, Hyderabad-500034 for  providing all the facilities

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