Exploring the Antiangiogenic and Anti-Inflammatory Potential of Homoisoflavonoids: Target Identification Using Biotin Probes

Abstract

Chemical proteomics using biotin probes of natural products have significantly advanced our understanding of molecular targets and therapeutic potential. This review highlights recent progress in the application of biotin probes of homoisoflavonoids for identifying binding proteins and elucidating mechanisms of action. Notably, homoisoflavonoids exhibit antiangiogenic, anti-inflammatory, and antidiabetic effects. A combination of biotin probes, pull-down assays, mass spectrometry, and molecular modeling has revealed how natural products and their derivatives interact with several proteins such as ferrochelatase (FECH), soluble epoxide hydrolase (sEH), inosine monophosphate dehydrogenase 2 (IMPDH2), phosphodiesterase 4 (PDE4), and deoxyhypusine hydroxylase (DOHH). These target identification approaches pave the way for new therapeutic avenues, especially in the fields of oncology and ophthalmology. Future research aimed at expanding the repertoire of target identification using biotin probes of homoisoflavonoids promises to further elucidate the complex mechanisms and develop new drug candidates.

Keywords: homoisoflavonoid; natural products; photoaffinity labeling; target identification.

Figure 1

Chemical proteomics to link phenotype-based drug discovery and target-based drug discovery.

Figure 2

Photoaffinity labeling for chemical proteomics. The asterisk (*) indicates the bands on the gel where the target protein is located. The red dots represent electrons generated by the photocrosslinker upon UV activation. These electrons form intermediates that readily bond covalently with cysteine residues near the binding pockets of the target protein and ligand, enhancing the compound’s interaction with the target protein.

Figure 3

(A) Comparative structures of homoisoflavonoid scaffold, flavonoid, and isoflavonoid; (B) biosynthetic pathway and structures of five types of homoisoflavonoids.

Figure 4

Design of biotin probes based on homoisoflavonoids.

Figure 5

Structure of cremastranone and its derivatives. (A) Cremastranone and design of homoisoflavonoid derivatives; (B) structure of antiangiogenic SH11008 and SH11037.

Figure 6

Identification of FECH as a target of the antiangiogenic natural product, cremastranone. (A) Chemical structures of cremastranone (1), control reagent (2), and photoaffinity probe (3). (B) Silver staining of SDS-PAGE-separated proteins from photoaffinity chromatography with indicated reagents showed specific bands at 43 and 35 kDa, which were excised and proteomically identified. The upper band was FECH. (C) Immunoblot of proteins that were isolated using an antibody against FECH. (D) Immunoblot of proteins extracted from a competition assay with excess active cremastranone isomer SH11052 (4). (E) Silver-stained SDS-PAGE gel of recombinant human FECH protein pulled down using photoaffinity chromatography. (F) Anti-FECH immunoblot of a similar pul-down experiment. Figure adapted from Figure 1 in Ref. [30].

Figure 7

Exploring the molecular target of cremastranone and developing FECH inhibitors.

Figure 8

Homoisoflavonoid analog SH11037 inhibition of sEH demonstrates its potential as an anti-angiogenic therapeutic agent. Figure adapted from Figure 1 in Ref. [34].

Figure 9

(A) Structures of SH11037-based photoaffinity probes. (B) Proteins isolated with indicated reagents were resolved by SDS-PAGE and silver stained, then identified by mass spectrometry. A distinct band was present in pull-down with 6 but not 5 or 7 (red box); asterisks represent nonspecific bands. MW in kDa indicated. Figure adapted from Figure 1 in Ref. [35].

Figure 10

(A) Structure of SA and its biotin probe. (B) Identification of SA target proteins using pull-down technology coupled with shotgun proteomics. (C) SA selectively binds to IMPDH2. Figure adapted from Figure 1 in Ref. [38].

Figure 11

(A) Structure of BZ and its biotin probe 10. (B) Identification of BZ target proteins using human proteome microarray. (C) DOHH was identified as the protein with the highest signal-to-noise ratio (SNR) when screening for BZ-binding proteins using a human proteome microarray. Figure adapted from Figure 2 in Ref. [44].