New allosteric inhibitor of AKT targets its PH domain
A significant goal in today’s drug discovery and biology research, particularly for therapies targeting cancer, is kinase function modulation. Most kinase inhibitors, known as ATP competitive, target active kinase ATP binding sites and impair their phosphorylation. A major issue with respect to these molecules is their lack of selectivity. A second class of small organic molecules that address remote kinase sites and stabilize enzymatically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. These inhibitors are better known as allosteric modulators, binding to sites that are less conserved across the kinome and stabilizing specific conformations of the enzyme. This article from Bill X. Huang is a typical case study for identifying such allosteric modulators.
An iconic kinase studied: AKT. An allosteric region of the kinase targeted: its PH domain. A robust and reliable assay using Neuro 2A cells: implemented. The author emphasizes the capability of transposing the assay from 384w to 1,536w, while maintaining excellent assay performance (Z’ Factor: 0.79, S/B: 7.4), and on signal stability for more than 24 hours. Altogether excellent assets for processing large numbers of HTS plates!
The mechanism of action for the most promising compound (G7) was further assessed using Western-blot and ELISA techniques. Selectivity, G7 potency, and effects on kinases involved either upstream of AKT or on downstream AKT signaling, are fully documented. Direct binding of G7 on the PH domain of AKT was further confirmed using microscale thermophoresis.
The exclusive structural features of the kinases’ PH domain and the possibility of modulating those features allosterically by chemical compounds open new doors in the quest for more specific kinase inhibitors.
Now, it’s time to set up your own HTS assay to identify new inhibitor classes of your favorite kinase!
Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.
: Scientific Reports, 2017 Sep 15;7(1):11673.