Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Summary

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

Abstract

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists¹. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Introduction

The GLP-1R is an established drug target in the treatment of type 2 diabetes². The native peptide agonist for this receptor, GLP-1, has an in vivo half-life of 2-3 min³. The binding of GLP-1 to its G protein coupled target receptor results in the downstream production of the second messenger cAMP through native G protein coupling to the activation of adenylyl cyclase. Measurement of the accumulated cAMP provides a robust assay to monitor receptor activation and to screen for active GLP-1 analogues with preferred physicochemical properties. Such an assay requires the serial dilution of test samples to construct concentration-response curv....

Protocol

1. Peptide Serial Dilution

1. Prepare assay buffer: Hanks buffered salt solution (HBSS) supplemented with 25 mM HEPES, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), pH 7.4.
2. Use a bulk reagent dispenser to systematically add 5 µl of assay buffer to each well of five 384-well low volume assay plates.
   1. Use internal software to create a dispensing program for 5 µl volume addition to every well of a 384-well plate as per manufacturer's instructions.
   2. Immerse dispensing cassette tubing in assay buffer and prime fluid.
   3. Place 384-well low volume assay plate on plate carrier.
   4. Press start.

Results

We routinely use a two-step method to dilute peptides via acoustic transfer. For the first step, an acoustic dispenser aligned with automation is used to create four stock peptide intermediate dilutions across two source plates (Figure 1a, b). For the second step, we use an acoustic dispenser to further dilute stock dilutions from source plates A and B to create an 11-point concentration range for each test peptide (Figure 1c). Each pepti.......

Discussion

This protocol describes the successful application of automated acoustic dispensing to serially dilute peptide samples over a concentration range of 3 x 10⁶ requiring less than 1 µl of sample. The major advantage of this method is to increase data quality through minimizing peptide adsorption to plasticware via reduced exposure of samples to experimental containers and plasticware (such as pipette tips) that are normally required for reagent transfer and mixing. While acoustic dispensing does not complete.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hanks’ Balanced Salt solution	Sigma-Aldrich	H8264
HEPES	Sigma-Aldrich	H3375
Bovine Serum Albumin	Sigma-Aldrich	A9418
3-Isobutyl-1-methylxanthine	Sigma-Aldrich	I7018	Prepared as a 0.5 M stock in DMSO
GLP-1 (7-36) amide	Bachem	H-6795	Prepared as a 1 mg/ml stock in PBS, referred to as '100x reference control'
Test peptides	Produced in-house at MedImmune		Supplied at various concentrations in DMSO or PBS as appropriate
100x peptide stock	Produced in-house at MedImmune		Test peptide diluted into assay buffer to 100x final required concentration
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250-061
Cedex XS Cell Analyzer	Innovatis
Corning 384 well plates, low volume	Sigma-Aldrich	4514
Echo Qualified 384-Well Polypropylene Microplate	Labcyte Inc.	P-05525
Echo Qualified Reservoir	Labcyte Inc.	ER-0055
Echo 550 Liquid Handler	Labcyte Inc.		Droplet transfer volumes in increments of 2.5 nl
Echo 525 Liquid Handler	Labcyte Inc.		Droplet transfer volumes in increments of 25 nl
ACell Benchtop Automation	HighRes Biosolutions	MC522
Cellario Lab Automation Scheduling software for Life Science Robotics	HighRes Biosolutions
MultidropCombi Reagent Dispenser	ThermFisher Scientific	5840300	Referred to as 'bulk reagent dispenser'
HTRF cAMP Dynamic 2 kit	Cisbio Bioassays	62AM4PEJ
EnVision Multilabel Reader	PerkinElmer