Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2

Abstract

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

Keywords: cancer; high-throughput screen; luminescence; lysyl hydroxylase-2 (LH2); succinate detection.

Figure 1.. LH2 assay optimization.

Assays were performed by varying A) LH2 concentrations (0 – 1 μM), B) [IKG]₃ peptide concentrations (0–1000 μM), and C) time (5, 60, 120 or 180 min; with (●) and without (■) LH2). D) A succinate standard curve fitted linearly (R² = 0.9988).

Figure 2.. Assay stability against additives or time.

Activity of LH2 (in %) were measured against variations of A) BSA, B) DMSO, C) triton X-100, D) tween-20, E) NP-40, and F) incubation time. In all cases, a positive control with 0% variation is normalized to 100 % activity. % activity of LH2 assays containing variations were calculated according to [$\frac{x_i}{x_0}\ast 100$], where x₀ is RLU for positive control and x_i is RLU for varying samples.

Figure 3.. Statistical analysis for the screen.

A) Scattered plot of z’ values for all assay plates. B) Histogram showing percent inhibition of LH2 activity against frequency of compounds (red, peak 1, 0% inhibition control; blue, peak 2, 100% inhibition control; green, peak 3, % inhibition by library compounds). C) Flowchart of hits identification and confirmation process.

Figure 4.. Dose Response activity of top 3 compounds against LH2.

X-axis represents different concentrations of compounds (in log[M]) whereas Y-axis represents inhibition of LH2 activity (in percent). In all cases, a no compound (0% inhibition) control was included and used as reference to calculate percent inhibition values. Percent inhibition was calculated according to equation (2) and data were fitted to four parameter logistic non-linear regression. Compounds 1 (●), 2 (■) and 3 (◆) show pIC₅₀ values of 6.94 ± 0.08, 6.52 ± 0.04 and 6.32 ± 0.10, respectively, where pIC₅₀ = −log(IC₅₀).