Molecular Interactions for In Vitro Proof-of-Concept Studies

Biochemical Characterisation of Inhibitor-Target Interaction for Precision Medicine

Selection of the best drug candidate for your target requires an understanding of the binding characteristics of the compounds to the target. Several biochemical techniques can be applied to select the compound with the best characteristics for further analysis.

Relating Drug Activity to Binding Characteristics via Biochemical Assays

The design of novel compounds with improved properties can be facilitated using a variety of assays. A typical workflow consists of 1. Protein expression and purification; 2. Development of an activity assay and selection of compounds; 3. Profiling compounds via SPR assay; 4. Thermal stability assay; 5. Crystal structure determination of the protein in the presence of promising inhibitors.

Protein Expression

1

Protein expression
and purification for use in biochemical assays

The protein of interest can be expressed in E. coli or the baculovirus expression system. Purification can be performed with different separation methods such as affinity chromatography, size exclusion, and anion exchange chromatography. Purity of the protein will be validated by SDS-PAGE analysis.

Screening Activity Assay

2 

Compound screening
in enzyme activity
assays

Different types of enzymatic assays can be developed in a 384-well format for screening a library of compounds. Readout of the assays can be based on absorbance, fluorescence, chemiluminescence, TR-FRET, or Alpha technology.
Mix-and-match assay technology that is available from Oncolines: Arginase Gold™ and NFK Green™.

Thermal Stability Assay

3

Profiling via SPR to measure the target residence time of drugs

The target protein can be immobilized on a Biacore chip to measure association and dissociation kinetics, and target residence time of compounds, via SPR. Single cycle kinetics and a 1:1 binding fit model are used to the determine the binding parameters.

Profiling Via SPR

4

Thermal stability assay to see the effect ofcompounds on stability of the protein

The melting (or denaturation) temperature of the protein is a measure of its thermal stability. An increase of the thermal stability of the protein in the presence of a compound is indicative of a (strong) protein-inhibitor interaction.

Protein Crystallisation

5 

Protein crystallisation
in the presence
of compound

Protein crystallization and crystal structure determination are used to identify the specific binding site and mode for the protein of interest. This can be achieved through co-crystallization of the protein in the presence of the compound or via soaking of preformed crystals in a solution of the compound.

  • Binding kinetics using surface plasmon resonance (SPR)

  • Biochemical potency through activity assays based on absorbance, fluorescence, chemiluminescence, TR-FRET, or Alpha technology

  • Protein stabilization via thermal stability assays

  • Inhibitor binding mode through crystallization of your target with inhibitors

  • ADME properties using various ADME assays for additional profiling of your inhibitor

Illustration Of The Development Of A Screening Assay
Fluorescence-based Arginase-1 activity assay
(A) Combined L-arginine and L-ornithine calibration curve at pH 9.5 and (B) pH 7.4, demonstrating that the fluorescent signal is linearly proportional to the L-arginine and L-ornithine concentrations within the measuring range of the assay.
(C) Time-course of Arginase-1 activity at different enzyme concentrations at pH 9.5 and (D) at pH 7.4 measured in real time. (Adapted from Grobben et al., 2020).

Deconvolution of Binding Mode, Site, and Mechanism

Our protein and biophysical scientists are in close contact with the sponsor to determine the most suitable assay format for the study and to share scientific insights. Oncolines also has expertise in cancer biology services. The biochemical and cell-based assay technologies provide a comprehensive portfolio for in vitro characterisation of your lead candidates.

Relation Between Cellular Activity And Dissociation Rate (via SPR) Of TTK Inhibitors
(A) Relation between cellular activity (on cell line MOLT-4) and dissociation rate (via SPR). The red circles are TTK reference inhibitors, the grey circles represent NTRC TTK inhibitor series.
(B) Comparison of association and dissociation rates for reference inhibitors. Diagonals represent equipotency lines with pertinent KD values indicated within the frame. The ATP-pocket binding (green, triangles) and lysine-targeting inhibitors (yellow, lozenge) can be distinguished as separate groups.
(C) Thermal unfolding curves of the TTK kinase domain in the presence of TTK inhibitors. Relatively fast dissociating compounds shift, unfolding to a lesser extent than slowly dissociating compounds. This indicates that the binding of TTK inhibitors with long target residence times induces a stabilizing conformational shift (Uitdehaag et al., 2017).
Representative Thermal Unfolding Curves Of The TTK Kinase Domain In The Presence Of TTK Inhibitors

In Vitro Studies Enhance Discovery of Drugs for Oncology Precision Medicine

It is our mission to help you find a mechanistic hypothesis before entering the clinic. We have generated science-based technologies that help you to identify the mechanism of preclinical and clinical drug candidates. Our technologies provide in vitro proof of concept in the preparation of in vivo studies and clinical trials.

Deliverables You Can Count On

We deliver reproducible results that are adapted to your specific needs with a short delivery time. Direct contacts between the sponsor and our staff result in efficient scientific and technical support. We provide free consultation on next steps in research if desired by the sponsor.

Quality. Flexibility. Short Turnaround Time.

References

Grobben et al. (2020) Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158. Journal of Structural Biology: X, 4:100014.

Grobben et al. (2020) High-throughput fluorescence-based activity assay for Arginase-1. SLAS Discovery, 25 (9):1018-1025.

Uitdehaag et al. (2017) Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity. Journal of Molecular Biology, 429:2211-2230.

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