Lesson 1 (Golden Rule): Don’t screen for direct PAK1-inhibitors in test tube!

This year a "Genentech" team developed a new series of PAK1-3 (so-called "group 1") inhibitors:

J Med Chem. 2015 Jun 1. [Epub ahead of print]

Structure-Guided Design of Group I Selective p21-Activated Kinase (PAK) Inhibitors.

Crawford JJ, Lee W, Aliagas I, Mathieu S, Hoeflich K, Zhou W, Wang W, Rouge L, Murray L, La H, Liu N, Fan PW, Cheong J, Heise C, Ramaswamy S, Mintzer R, Liu Y, Chao Q, Rudolph J.

Abstract

Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs. II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.

The IC50 (cell permeability) of a new PAK1-3 inhibitor (called compound 23) in cell cuture is around 120 nM, similar to a herbal PAK1-blocker called "cucurbitacin I" from an edible bitter melon grown in Okinawa. We shall see its "bioavailability" and "safety" in vivo (animal test).

The direct PAK1-inhibitors called FRAX486 and FRAX597 developed by AFRAXIS (founded by Susumu Tonegawa of MIT, the 1987 Nobel laureate) have the IC50 around 10 nM for inhibiting PAK1 in test tube. However, their IC50 for inhibiting the PAK1-dependent growth of tumor cells in cell culture is above 1 micro M (1000 nM), clearly indicating that they are "very poorly cell-permeable", and therefore not useful for clinical application. In fact,  a recent in vivo study confirmed that FRAX597 (3 mg/kg, i.p.) alone has no effect on the growth of human pancreatic cancer xenograft in mice, while a herbal PAK1-blocker called glaucarubinone (1-2 mg/kg, i.p.) inhibits the growth of the same cancer xenograft by 70%.   

Again, a Novartis team recently developed a new PAK1-specific compound 3, which inhibits PAK1 with the IC50 around 10 nM in test tube, but in cell culture its IC50 is around 2 micro M (2000 nM), clearly indicating that compound 3 is no good for clinical application. So have you got any lesson?