Select azo compounds post-translationally modulate HTRA1 abundance and activity potentially through interactions at the trimer interface

Abstract

High-temperature requirement protein A1 (HTRA1) is a secreted serine protease with diverse substrates, including extracellular matrix proteins, proteins involved in amyloid deposition, and growth factors. Accordingly, HTRA1 has been implicated in a variety of neurodegenerative diseases including a leading cause of blindness in the elderly, age-related macular degeneration (AMD). In fact, genome wide association studies have identified that the 10q26 locus which contains HTRA1 confers the strongest genetic risk factor for AMD. A recent study has suggested that AMD-associated risk alleles in HTRA1 correlate with a significant age-related defect in HTRA1 synthesis in the retinal pigmented epithelium (RPE) within the eye, possibly accounting for AMD susceptibility. Thus, we sought to identify small molecule enhancers of HTRA1 transcription and/or protein abundance using an unbiased high-throughput screening approach. To accomplish this goal, we used CRISPR/Sp.Cas9 engineering to introduce an 11 amino acid luminescent peptide tag (HiBiT) onto the C-terminus of HTRA1 in immortalized ARPE-19 cells. Editing was very efficient (~88%), verified by genomic DNA analysis, short interfering RNA (siRNA), and HiBiT blotting. Nineteen-hundred and twenty compounds from two libraries were screened. An azo compound with reported anti-amyloidogenic and cardioprotective activity, Chicago Sky Blue 6B (CSB), was identified as an enhancer of endogenous HTRA1 secretion (2.0 ± 0.3 fold) and intracellular levels (1.7 ± 0.2 fold). These results were counter-screened using HiBiT complement factor H (CFH) edited ARPE-19 cells, verified using HiBiT blotting, and were not due to HTRA1 transcriptional changes. Importantly, serine hydrolase activity-based protein profiling (SH-ABPP) demonstrated that CSB does not affect HTRA1's specific activity. However, interestingly, in follow-up studies, Congo Red, another azo compound structurally similar to CSB, also substantially increased intracellular HTRA1 levels (up to 3.6 ± 0.3 fold) but was found to significantly impair HTRA1 enzymatic reactivity (0.45 ± 0.07 fold). Computational modeling of potential azo dye interaction with HTRA1 suggests that CSB and Congo Red can bind to the non-catalytic face of the trimer interface but with different orientation tolerances and interaction energies. These studies identify select azo dyes as HTRA1 chemical probes which may serve as starting points for future HTRA1-centered small molecule therapeutics.

Figure 1.

Design and validation of HTRA1 HiBiT editing strategy in ARPE-19 cells. (A) HTRA1 genomic DNA sequence and design of a C-terminal HiBiT HA insertion. (B) HTRA1 exon 9 gDNA amplification of unedited and edited ARPE-19 cells. Insertion of the VS HiBiT GG HA sequence increases the predicted molecular weight by 72 bp. Estimated editing efficiency (top band/bottom band) is ~88%. (C) Validation of HiBiT insertion at the HTRA1 locus by siRNA. ARPE-19 HTRA1 HiBiT edited cells were reverse transfected with control siRNAs (non-targeting, siTOX), or siRNAs against HTRA1, HTRA2, or HTRA3. n = 4 independent experiments for HTRA1 siRNAs, n = 2 independent experiments for HTRA2 and HTRA3 siRNAs. n.s. = not significant, **** p < 0.0001, one-way ANOVA with multiple comparisons vs. non-targeting. (D) HTRA1 HiBiT protein migrates at the expected molecular weight of ~55 kDa. ARPE-19 HTRA1 HiBiT cells were incubated in serum free media followed by concentration and analysis by HiBiT blotting. n = 3 independent experiments. (E) Nic differentiation media increases HTRA1 secretion. ARPE-19 HTRA1 HiBiT cells were plated at confluence followed by incubation in either full DMEM/F12 or Nic media for up to 7 days. A HiBiT assay was performed on media aliquots for 1 week. n = 3 independent experiments, **** p < 0.0001, unpaired t-test with multiple comparisons vs. respective DMEM/F12 control.

Figure 2.

Summary of HTS primary screen and dose-response. (A) An example HTS assay plate from the primary screen of the NIH and Prestwick libraries. Three standard deviations of the mean is shown by the dotted green line whereas three standard deviations below the mean is shown by the dashed red line. Compounds outside of these lines were considered hit compounds. (B) Top ten compounds from a list of 17 small molecules identified in the primary screen. (C) Reproduced dose-responsiveness of two hit compounds, cefixime trihydrate and Chicago Sky Blue 6B.

Figure 3.

Confirmatory studies on the effect of Chicago Sky Blue 6B (CSB) on HTRA1 HiBiT produced from ARPE-19 cells. (A) Azo-based chemical structure of CSB. (B) Validation of the effects of CSB and deconvolution of the previous whole well results into extracellular and intracellular components. ARPE-19 HTRA1 HiBiT cells (grown in Nic media) were treated with the indicated concentration of CSB for 72 h. n = 3–4 independent experiments, n.s. = not significant, ** p < 0.01, *** p < 0.001, one-way ANOVA with multiple comparisons vs. DMSO. (C) CSB increases HTRA1 HiBiT protein levels intracellularly and extracellularly as determined by a HiBiT assay. Cells were treated with 20 M CSB for 72 h in serum free media followed by concentration of the conditioned media, lysis of the cells and HiBiT blotting. Representative data of three independent experiments. (D) CSB does not alter HTRA1 expression levels. ARPE-19 cells were treated with 20 M CSB for 72 h in Nic media followed by mRNA extraction and qPCR. Representative data of three independent experiments. (E) CSB has no effect on HiBiT CFH secretion or intracellular levels. A control ARPE-19 cell line expressing HiBiT CFH (Sup. Fig. 2) was treated with CSB at the indicated concentration for 72 h, followed by HiBiT assay. n = 3 independent experiments n.s. = not significant, one-way ANOVA with multiple comparisons vs. DMSO.

Figure 4.

Additional azo dyes affect HTRA1 levels. (A, B) Chemical structures of azo dyes Congo Red (Congo R) and Brilliant Yellow (Bril Y). (C) Congo R, but not Bril Y increases HTRA1 intracellular levels. ARPE-19 HTRA1 HiBiT cells grown in DMEM/F12 full media were treated with the indicated compound (20 M) for 72 h followed by a HiBiT assay. Representative data of 3 independent experiments. n.s. = not significant, *** p < 0.001, **** p < 0.0001, one-way ANOVA with multiple comparisons vs. DMSO control. Note: Congo R data were compared to a different DMSO control than presented. (D) Azo dyes similarly alter CMV-driven HTRA1 HiBiT levels. ARPE-19 cells expressing an overexpression plasmid encoding for HTRA1 HiBiT 6xHis were treated as described in (C) followed by a HiBiT assay. Representative data of 3 independent experiments. n.s. = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with multiple comparisons vs. DMSO control. (E, F) HTRA1 variants with mutations near the dimer interface are affected by CSB differently than WT HTRA1. (E) Stable ARPE-19 cells expressing WT or R166C HTRA1 HiBiT 6xHis were treated with CSB for 72 h followed by a HiBiT assay of (E) extracellular or (F) intracellular HTRA1. Data pooled from three independent experiments. n.s. = not significant, ** p < 0.01, two sided t-test.

Figure 5.

Serine hydrolase activity-based protein profiling (SH-ABPP) post azo compound treatment. (A) Congo R inhibits human recombinant HTRA1 protein activity. HTRA1 protein was incubated with 20 mM of compound for 1 h followed by SH-ABPP using a FP-TAMRA probe. (B) FP-TAMRA signal was normalized to total protein (Coomassie). Representative data shown of at least two independent experiments performed in technical duplicate/triplicate. n.s. = not significant, **** p < 0.0001, one-way ANOVA with multiple comparison test vs. DMSO control. (C, D) Whole cell SH-APBB of ARPE-19 (endogenous) HTRA1 HiBiT cells treated with azo dyes (20 M, 72 h) and the corresponding total protein stain (Coomassie). Representative data of three independent experiments. (E) Quantification of total TAMRA signal across all experiments and normalized to DMSO controls. n.s. = not significant, one-way ANOVA.

Figure 6.

Compound binding to HTRA1 simulation. (A) Workflow for modeling interactions between azo compounds and HTRA1 models. (B-D) Binding site models of the top 5 scoring ligand positions, surface representation (top). 2D histogram of the sampled ligand positions (aspect ratio) and interface scores for CSB (B) Congo R (C) and Bril Y (D) colored from gray to blue based on the sampling frequency (bottom). A lower aspect ratio indicates less tolerated movements of the molecule within the model, whereas a higher interface score indicates Binding site models highlighting interactions between ligand the trimeric HTRA1 and CSB (E), Congo R (F) and Bril Y (G). HTRA is shown in cartoon representation. Interacting residues are shown in surface and stick representation. CSB, Congo R and Bril Y are shown in sticks and colored blue, red and yellow, respectively. Residues interacting in each model are labelled. (H-J) Summary of cumulative ligand residue contacts across modeled ensembles between HTRA1 and CSB (H), Congo R (I) and Bril Y(J). Residue contacts to ligand are shown for each HTRA1 chain in the trimer defined as A, B and C.