Cassia obtusifolia MetE as a Cytosolic Target for Potassium Isolespedezate, a Leaf‐Opening Factor of Cassia plants: Target Exploration by a Compact Molecular‐Probe Strategy

Abstract

Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

Functional importance of the sugar moiety of jasmonic acid glucoside for bioactivity and target affinity

“…In both the alkyne units, the FLAG peptide was conjugated as a molecular tag. 19,26 As shown in Fig. S1, † CuAAC using 7 gave the better result.…”

“…We synthesized 1, ent-1, 2 (Scheme S1 †), ent-2 (Scheme S2 †), and 3 according to the procedure in ref. 19. The open-chain type 4 was synthesized as shown in Scheme S3.…”

Chemical Biology of Natural Products on the Basis of Identification of Target Proteins

“…Recent progress in chemical biology brings us some promising methods applicable for target ID of NOLs, such as FG-beads technology, 26 photoaffinity beads technology, 27 a fishing-rod strategy, 28 a "compact" molecular probe (CMP) approach, 29 the yeast 3-hybrid (Y3H) system, 30 and a database-dependent (DBD) strategy 31,32 ( Figure 4). Historical background of these techniques can be found in excellent reviews.…”

“…The CMP approach provided a perfect result in which only the specific target was tagged without any nonspecific binding. 29 These successful results were obtained in the target IDs of isolespedezic acid and jasmonate glucoside. A similar concept employing a unique double-click strategy was also developed by Hosoya.…”

“…The CMP approach 29 developed by Ueda and co-workers ( Figure 4D) is a state-of-the-art technology for the target ID of NOLs. This concept is based on the idea that the NOL itself has the best affinity with its target and any modification of a functional probe will decrease its original affinity.…”