Discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of AKT/PKB: mechanistic involvement of AKT catalytic activation loop cysteines

Abstract

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC 50 , 0.149 F 0.045 Mmol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors. [Mol Cancer Ther 2007;6(11):3028 -38]

Drug discovery approaches targeting the PI3K/Akt pathway in cancer

“…Allosteric inhibition of Akt has also been reported for lactoquinomycin (compound 29, Figure 4) (Toral-Barza et al, 2007), a natural product isolated from the fermentation broth of a microbial strain Streptomycin sp. LL-AF101.…”

Critical and diverse involvement of Akt/mammalian target of rapamycin signaling in human lung carcinomas

“…In this sense, combined treatments with rapamycin and Akt inhibitors, such as lactoquinomycin and KP372-1, could be novel regimens. 33,35,36 The current results could provide a rationale for tailoring combination therapies for lung cancer, especially AC, using inhibitors of EGFR signaling and rapamycin. 2 In conclusion, the current study on lung carcinomas provides a detailed description of the proteins downstream of EGFR, in particular, those in the mTOR pathway, and the results have several clinicopathologic implications.…”

Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin

“…For protein synthesis assays, methionine incorporation (22), 7-methyl-GTP pull-down (23), and polysome analysis (22) were performed (Supplementary Methods).…”