Lesson 2: Start with a "highly cell-permeable" ATP antagonist to develop "potent" PAK1-blockers

Back in 2001, we identified the first potent PAK1-inhibitor among "marine" derivatives of staurosporine (ST) called "ST-2001" (or 3-OH ST) in which the position 3 of ST is hydroxylated. Its IC50 for both PAK1 in test tube and anti-cancer activity in cell culture is around 1 nM, indicating that its "cell-permeability" is very high. Unfortunately, however, it turned out to be a "non-specific" kinase inhibitor and "toxic" for normal cells as well.

The best (and probably simplest) way to make this marine compound "PAK1-specific" would be to link "bulky" and "positively charged" side chains (such as hexylamine) to the position 9 by an enzymatic synthesis of ST-3009. This modification would unable many kinases such as PKC (essential for the growth of normal cells) to accomodate this bulky compound in their relatively small ATP-binding pocket, except for PAK1 (non-essential for the growth of normal cells). PAK1 forms an inactive homodimer whose ATP-binding pocket is larger than any other kinases.

The major set-back of this project is that the marine source of this compound suddenly vanished from the coast line of Guam Island, perhaps due to a global warming (a rise in sea temperature) . On the other hand, chemical hydroxylation of ST only at position 3 (or 9) is possible, but "economically unfeasable" (the yield is very low!).  Thus, as an alternative, we search for an enzyme (called ST hydroxylase, STOHase) which is able to hydroxylate ST at either position 3 or 9 (but not both positions!).

Currently, we are screening for extracts from marine organisms such as sea cucumber which contain PAK1-blockers and would be able to convert ST to ST-2001 in vitro. Once ST-2001 is produced in test tube, its chemical modification at position 9 would be a piece of cake. In the past we have learned lots of lesson from CEP-1347 (1).  Recently we learned another key lesson from the enzymatic synthesis of ARC (artepillin C) using a gio-specific yeast lipase B (Novozym 435) which could distinguish the ester bond at position 3 from that at 9 of ST (2).

References:

1. Nheu, T., He, H., Hirokawa, Y., Tamaki, K. et al. The K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS transformants. Cancer J. 2002, 8, 328-36.

２．Yashiro K, Hanaya K, Shoji M, Sugai T. New synthesis of artepillin C, a prenylated phenol, utilizing lipase-catalyzed regioselective deacetylation as the key step. Biosci Biotechnol Biochem. 2015、 18:　1-5.