Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation

Abstract

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog-glioma (Hh-Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein-protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine-indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell-based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α-tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α-tubulin from its stabilised form.

Evaluation of a Tubulin‐Targeted Pyrimidine Indole Hybrid Molecule as an Anticancer Agent

“…Our earlier report [19] suggested that substitution can be incorporated very easily at 1 or 2-position of indole in PIH (P) without changing much in the biological activity. Accordingly, for the synthesis of water soluble derivatives of PIH (P) we then decided to incorporate water soluble moiety in the indole part at this position keeping the hydrazine part of the molecule intact.…”

“…For the synthesis of N-alkylated indole derivative the reaction was started from 2methylindole-3-carboxaldehyde (3 B). [19] The indole was alkylated with triethylene glycol 2-bromoethyl methyl ether to get N-alkylated indole derivative (4 C). These three indole derivatives were reacted with hydrazine derivative to get the final compounds 6 A, 6 B and 6 C. In accordance with our earlier report, [19] all the compounds were characterized by spectroscopic methods and found to be single-isomers with the appearance of a single set of peaks in 1 HNMR.…”

“…[19] The indole was alkylated with triethylene glycol 2-bromoethyl methyl ether to get N-alkylated indole derivative (4 C). These three indole derivatives were reacted with hydrazine derivative to get the final compounds 6 A, 6 B and 6 C. In accordance with our earlier report, [19] all the compounds were characterized by spectroscopic methods and found to be single-isomers with the appearance of a single set of peaks in 1 HNMR. Comparing with the previously reported hydrazone derivatives of E-conformers for antiproliferative activity, [21] we believed the most stable Eisomer was formed in our case.…”

“…[15][16][17][18] In our laboratory, the mechanism of action of a small molecule based on pyrimidine indole hybrid (PIH (P)) structure was delineated and found to inhibit ciliogenesis by inhibiting the polymerization of tubulin subunits. [19] This result encouraged us to study the anticancer activity of PIH (P). For any drug to achieve the required pharmacological response, one of the important parameters to be taken into consideration is its solubility in water.…”

Design of Hedgehog pathway inhibitors for cancer treatment

“…Inspired from the interesting Hh signaling antagonist potency of pyrimidine-indole hybrid (PIH) structure, 54 Khatra et al 55 have designed a small compound library of PIH series 3k (Figure 3B) and identified their distinct modes of action as Hh inhibitors. All the new compounds were screened in Shh-Light 2 cells at 1.0 mM concentration.…”