A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry

Abstract

The rapid dilution of the enzyme-inhibitor complex assay to monitor the recovery of enzyme activity is a well-established assay to determine the reversibility of inhibition. Our laboratory has previously employed this method to ascertain the reversibility of known glutamate carboxypeptidase II (GCPII)-targeting agents. Due to the tedious and time-consuming nature of the assay, we sought to develop a facile method to determine the reversibility of well-characterized GCPII inhibitors using bio-layer interferometry (BLI). The results from the BLI assay are in agreement with the rapid dilution method. Herein, we report for the first time, a rapid, novel real-time BLI method to determine reversibility of inhibition.

• BLI
• GCPII
• PSMA

Introduction

The expression of glutamate carboxypeptidase II (GCP II) in human prostate epithelium is known as prostate-specific membrane antigen (PSMA). GCP II is a glycosylated cell-surface zinc metallopeptidase uniquely expressed on prostate tumor cells and the neovasculature of non-prostatic malignancies1^,2. As a consequence, GCP II has attracted significant attention over the past several years as a target for the delivery of imaging and therapeutic agents, and continues to serve as an important clinically-relevant biomarker.

GCP II is reported to possess two predominant, yet poorly understood, enzymatic activities: the hydrolytic cleavage and liberation of glutamate from γ-glutamyl derivatives of folates3 and the proteolysis of the neuropeptide N-acetylaspartylglutamate (NAAG)4. Although its role in the progression of prostate cancer remains conjectural, there is emerging evidence that GCP II plays a regulatory role in angiogenesis5. Various chemical scaffolds have been developed as inhibitors of this enzyme to selectively deliver imaging and therapeutic agents6–8.

We previously reported a series of phosphoramidate peptidomimetic inhibitors of PMSA and classified their reversibility of inhibition by monitoring the recovery of enzyme activity following rapid dilution of the enzyme-inhibitor complex9. In addition, the correlation between reversibility of inhibition and GCP II internalization in LNCaP cells has been determined; we found that pseudo-irreversible inhibitors induced internalization to a greater extent than slowly reversible or reversible inhibitors9. Furthermore, we recently demonstrated that reversibility of inhibition has an effect on internalization and percent uptake of GCP II-targeted SPECT agents with the irreversible-targeting agent, demonstrating superior uptake and internalization in GCP II-positive (GCP II+) cells10. These results confirm the significance of pseudo-irreversible inhibitors as targeting molecules in the development of targeted imaging and therapeutic agents and provide rationale for establishing a convenient and rapid method for ascertaining mode of inhibition. The focus of the work described herein was aimed at developing an efficient method to determine the mode of inhibition for inhibitors of GCP II; our current benchmark method (a rapid dilution, HPLC-based assay) is tedious9. An attractive aspect of a bio-layer interferometry (BLI)-based assay is the small quantities of reagents required, which can also be reused for subsequent assays. BLI also allows for real-time monitoring of on- and off-rates of small enzyme-small molecule interactions. In addition, multiple inhibitors can be evaluated simultaneously for dissociation constants and reversibility of inhibition.

In prior studies, we reported the IC₅₀ values and reversibility of inhibition for CTT54 (1, IC₅₀=14 nM)11 and CTT54.2 (2, IC₅₀=144 nM)12 (Figure 1), utilizing a commonly employed method which involves monitoring the recovery of enzyme activity following rapid dilution of the enzyme-inhibitor complex by HPLC9. The results from these rapid dilution assays were used to validate the real-time BLI assay employed in this study as a qualitative method to assess the mode of inhibition.

Figure 1. Structures of known GCPII inhibitors and biotinylated analogs.

Establishing a BLI-based binding assay for GCP II required baiting a BLI biosensor tip with a small molecule, which inhibits the enzymatic activity of GCP II, in order to capture GCP II from a buffered solution. We chose to prepare biotinylated derivatives of known GCP II inhibitors linked through a PEG₁₂ linker; we previously found biotinylated GCP II inhibitor with a PEG linker did not alter the reversibility of inhibition13. CTT54 (1), CTT54.2 (2), and biotin-PEG₁₂-CTT-54 (3) were available from previous studies12^,13. Biotin-PEG₁₂-CTT54.2 (4) and Biotin-PEG₁₂-Lys-Urea-Glu (6) were prepared from compounds 2 and 5 (see Supplementary material for synthesis) and biotin-PEG₁₂-NHS using the same protocol to prepare biotin-PEG₁₂-CTT-5414.

Methods and material

BLI assay

The reversibility of inhibition studies (pseudo-irreversible, slowly reversible, and rapidly reversible) for the biotinylated derivatives (3, 4, and 6) of known GCPII inhibitors on the Octet Red (ForteBio, Menlo Park, CA) system were all carried out in 96-well plates (Greiner) at 30 °C and shaken at 1000 rpm for each step. For each reversibility of inhibition experiment, compound 3, 4, or 6 (100 μM, 250 μL), were added to individual wells of a 96-well plate. Streptavidin-coated biosensor tips (ForteBio) were mounted in the instrument and dipped into respective wells containing compound 3, 4, or 6 and incubated for 600 s to saturate the tips with each compound. Unoccupied streptavidin binding-sites were blocked by incubating the loaded biosensor tips in wells containing biocytin (1 mM, 250 μL) for 600 s. Unbound biocytin was then removed by dipping and incubating the biosensor tips in well containing Tris buffer (250 μL, 50 mM) for 600 s. Purified GCP II15 was then loaded onto the biosensor tips by dipping and incubating the inhibitor-coated tips in wells containing a solution of the protein (25 μg/mL, 250 μL) for 600 s. To obtain a clean dissociation signal, the biosensor tips loaded with the GCP II-inhibitor complex were then dipped into wells with 50 mM Tris buffer (250 μL, 50 mM) containing 1% triton X-100 and compound 1 (14 μM; 1000-fold greater than the IC₅₀ value of 1) for 28 800 s.

Results and discussions

The dissociation of the GCP II-inhibitor complexes with 3, 4 and 6 was observed for 8 h. In the case of reversible inhibitors, a rapid exponential decay curve would be observed for the dissociation of the enzyme-inhibitor complex16^,17. For an irreversible inhibitor, the enzyme-inhibitor complex dissociation progress curve should be flat except for instrumental drift. The progress curve for a slowly reversible inhibitor in the time scale of the activity assay, should be similar to that of a reversible competitive inhibitor but with a slower decay. The observed dissociation curves for the biotinylated derivatives 3, 4 and 6 were consistent with the known modes of binding for compounds 1,11 2,12 5, and their biotinylated derivatives 3, 4, and 6. Compound 3 showed little or no change in the experimental time frame while GCP II dissociated slowly from the enzyme-inhibitor complex with 4 (Figure 2). These observations are distinct from what is normally observed for classical competitive inhibitors16^,17. On the other hand, compound 6 showed a rapid exponential decay, which is in agreement with the results from the HPLC assay that the urea compounds 5 and 6 are reversible.

Figure 2. Dissociation of the irreversible GCP II inhibitor 3 (Biotin-PEG₁₂-CTT54), slowly-reversible inhibitor 4 (Biotin-PEG₁₂-CTT54.2), and reversible inhibitor 6 (Biotin-PEG₁₂-Lys-Glu urea), compound structures adjacent to the curves, in (50 mM TRIS, pH 7.4 with 1% X-100 trition) buffer containing 14 μM CTT54 (1).

Conclusion

These results support the validation of BLI as a qualitative method to rapidly assess the reversibility of inhibition in real-time, which should be broadly applicable to various small-molecule enzyme inhibitors and receptor ligands. We anticipate the development of this BLI method will allow our lab and others to rapidly identify pseudo-irreversible inhibitors, and to advance them as targeting molecule candidates for both imaging and therapeutic applications for prostate cancer. To the best of our knowledge, this is the first BLI assay developed to qualitatively identify reversibility of inhibition. Subsequent studies will be aimed at developing a more universal BLI assay to determine mode of inhibition, k_on/k_off rates, or IC₅₀ values without the need to biotinylate each compound of interest.

Supplementary material available online

Acknowledgements

The authors extend their gratitude to both Prof. Jeffrey P. Jones for access to the OctetRED system and ForteBio for technical assistance.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported in part by the National Institutes of Health (R01CA140617).