Screening of crude reaction mixtures

Introduction

Fragment screening has emerged as a powerful approach for identifying initial hit compounds in the drug discovery process. The central feature is that a small number (1000s) of low molecular weight (typically <250 Da) compounds are screened for binding to a target. The small size of the compounds means they are more likely to bind, but with weak affinity, and many different biophysical approaches can be used, including Creoptix sensors. It is relatively straightforward to identify fragments that bind to most binding sites on most targets. The challenge is growing the fragments to larger hit compounds with higher affinity before beginning to optimize for drug properties.

The affinity for a compound (analyte, A) binding to a target (ligand, L) assumes a simple equilibrium as:

where k_d is the dissociation or off-rate and k_a the association or on-rate and the equilibrium dissociation constant or affinity (K_D) is the ratio k_d / k_a. In many cases, improvements in affinity occur when modifications to the compound result in a slower off-rate. This is a first-order rate constant and is thus independent of concentration.

Conventional approaches to optimization require individual reactions to synthesize each compound, followed by purification and making up the compound at a defined concentration before measuring the affinity in an assay. Vernalis has pioneered an approach where crude-reaction mixtures (CRM) are screened which dramatically improves the speed and cost of synthesis to explore opportunities for improving compound affinity. For a hit compound, a set of reactions are performed in parallel usually in a plate-based format, changing the substituent(s) incorporated into the hit compound. After minimal workup, the resulting crude reaction mixtures are then individually assessed for changes in the off-rate for binding to the target – a change in off-rate indicates that a compound of improved affinity has been obtained. The initial demonstration of such off-rate screening (ORS)¹ used Biacore technology to demonstrate efficiency improvements in terms of materials, associated waste disposal and time. In addition, more compounds can be ranked rapidly in the early stages of a project, when understanding of structure-activity relationships (SAR) is potentially low.

The ability to reliably detect changes in off-rate is key to this approach. The proprietary Grating-Coupled Interferometry (GCI) technology delivers superior sensitivity over traditional technologies, such as Surface Plasmon Resonance (SPR), allowing reliably determination of off-rates of up to 10s^-1. In addition, the no-clog microfluidics of the WAVEsystem, a Creoptix technology, allow a wide variety of solvents, including acetonitrile and high concentrations of DMSO, increasing the range of chemistries that can be used in crude reaction mixtures.

In this technical note, we have compared the ORS results obtained with the WAVEsystem to those previously obtained by Vernalis with the Biacore instrument, retrospectively using the same library of CRMs against two targets, the Pyruvate Dehydrogenase Kinase 2 (aa16-407), hereafter referred to as PDHK2 and the  N-terminal ATPase domain of heat shock protein 90α (aa9–236), hereafter referred to as Hsp90. Vernalis has previously identified a series of potent inhibitors for both of those members of the GHKL family of ATPases.^2,3,4

Materials and Methods

Off-rate screening of crude reaction mixtures (CRM). Eighty-three (83) CRMs and a purified control compound were screened for their off-rates on the WAVEdelta. A PCH-NTA WAVEchip was loaded with nickel chloride prior to capturing the target proteins. Double His6-tagged HSP90 and double His6-tagged PDHK2  proteins were then captured at ca. 7000 pg/mm² onto channels 2 and 3 respectively. Channels 1 and 4 were nickel-loaded and were used as reference channels. The CRMs were injected at approximately 20 μM concentration in HBS-P +1%DMSO for 30s at a constant flow rate of 250 μl/min. Dissociation was allowed for 120s. Offrate determination was performed using the WAVEcontrol software.

Purified compound kinetics

A PCH-NTA WAVEchip was loaded with 0.5 mM NiCl² prior to the capture of the proteins. HSP90 and PDHK2 were captured in HBS-P buffer at a density of ca. 3500 pg/mm2. VER235377 (purified compound) was injected at 30 μl/min in HBS-P + 1%DMSO at increasing concentrations ranging from 27.4 nM to 20 μM (7 concentrations, 3-fold dilutions). Injection was allowed for 60s prior to a 60-second dissociation phase. All interaction analysis was performed at 25°C and data was evaluated using the WAVEcontrol. A Langmuir 1:1 model was used for data fitting and kinetic parameters were determined.

Results

Vernalis has pioneered the use of off-rate screening (ORS) to kinetically sample in the hit-to-lead chemical space.¹ Their expertise in cheminformatics, compound library synthesis and Surface Plasmon Resonance (SPR) analysis, was attractive for this benchmark study and enabled the ORS of unpurified reaction products (CRM). CRM screening rapidly identifies lead compounds from fragment hits without purification of compound libraries or the use of protein structure. A comparative ORS study was conducted using the WAVEdelta (based on the GCI technology) and the Biacore T200 (based on the SPR technology) to compare and contrast the two technologies. Specifically, selected compounds were measured on the WAVEdelta and compared to a retrospectively tested (on a Biacore T200) CRM library from Vernalis. 

Figure 1 shows off-rate determination examples of selected CRMs. In both experiments, the results obtained from WAVEdelta were in agreement with those obtained from the Biacore T200. For those compounds with off-rates above 1s^-1, the WAVEdelta was able to reliably resolve those very fast dissociation constants (k_off).

Figure 1: Off-rate screen of selected CRMs. Measurements were done either on a Biacore T200 (left panel) or Creoptix WAVEdelta (right panel). GCI data reproduces the SPR data and enables an accurate and more reliable off rate determination of interactions faster than 1s^-1

This off-rate screen study led to the identification of the same PDHK2-selective hit that was also characterized as purified compound (VER235377) on the WAVEdelta. Kinetics parameters and binding affinity were similar to those obtained by the SPR-based Biacore T200. Figure 2 shows data obtained by GCI WAVEdelta instrument, right panels) and SPR (Biacore T200, left panels). Finally, we identified the same PDHK2-selective hit compound (VER235377).^2,4 The kinetic data obtained is shown in Table 1.

	kon (M-1.s-1)	koff (s-1)	Rmax (pg/mm2)	KD (nM)
HSP90 Biacore T200	1.92x105	0.130	24.1	679
HSP90 WAVEdelta	1.82x105	0.122	25.2	669
PDHK2 Biacore T200	6.10x105	0.116	18.3	191
PDHK2 WAVEdelta	3.16x105	0.052	14.5	166

Table 1: Kinetic data for Tpurified compound VER235377 over HSP90 and PDHK2

Figure 2: Binding kinetics data of purified compound VER235377. Measurements were done either on a Biacore T200 (left panel) or WAVEdelta (right panel). Comparable  data were obtained for both proteins HSP90 and PDHK2

In short, with the Creoptix WAVEsystem we have been able to:

• Confirm previously reported and measured with a Biacore T200 PDHK2 hit^2,4
• Confirm the same PDHK2-selective hit VER235377 and characterize it in its fully purified form
• Confirm VER235377 PDHK2 selectivity over HSP90
• Show excellent resolution for compounds with off-rates well above 1s^-1, highlighting the ability of the WAVEsystem to resolve fast off-rates

Conclusion

The Creoptix WAVEsystem offers kinetic data in excellent agreement with Biacore T200 measurements, as shown by the results obtained for a retrospectively measured Vernalis compound campaign against Pyruvate Dehydrogenase Kinase 2 (PDHK2) and Heat Shock Protein 90 (HSP90). With high sensitivity, the ability to resolve extremely fast interactions, the WAVE improves compound screening and kinetic analysis of small molecules to accelerate drug development and significantly reduce the costs associated with target and compound purification. The WAVE is therefore ideally placed to screen libraries of weak binders that display very fast off-rates such as small molecules, fragments, peptides, etc. Combined with a no-clog microfluidic technology that enables the study of crude, unpurified reaction mixtures, the WAVE is revolutionizing and speeding up the drug discovery process.

Key takeaways

Capture fast off-rate of weakly binding compounds in crude reaction mixtures with the WAVEsystem, a Creoptix technology.

• No clog microfluidics compatible with crude reaction mixtures: ideal for off-rate screening and fragment-based drug design
• Data comparable with SPR historical data: GCI technology delivers similar kinetic data to that obtained with SPR-based instrumentation
• Well resolved off-rates above 1 s^-1: with fast transition, the WAVEsystem measures off-rates of up to 10 s^-1

Great for:

• Off-rate screening of crude mixtures: small molecules, fragments, and peptides
• Fast hit-to-lead progression
• Lead optimization

References

1. Murray, J.B. et al. 2014. Off-Rate Screening (ORS) By Surface Plasmon Resonance. An efficient method to kinetically sample hit to lead chemical space in unpurified reaction products. J. Med. Chem. 57, 2845-1850 doi: /10.1021/jm401848a
2. Brough, P.A. et al. 2017. Application of Off-Rate Screening in the Identification of Novel Pan-Isoform Inhibitors of Pyruvate Dehydrogenase Kinase.J. Med. Chem., 60, 6, 2271–2286. doi: 10.1021/acs.jmedchem.6b01478
3. Brough, P. A. et al. 2009. Combining hit identification strategies: fragment-based and in silico approaches to orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone. J. Med. Chem. 52, 4794–4809 doi: 10.1021/jm900357y
4. Baker, L .M. et al. Rapid optimization of fragments and hits to lead compounds from screening of crude reaction mixtures. Commun Chem 3, 122 (2020).doi: 10.1038/s42004-020-00367-0

Acknowledgments

We thank Dr. Natalia Matassova (Vernalis) for carrying out all experiments and Prof. Roderick Hubbard for their contribution to this technical note.

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