Identification and development of a subtype-selective allosteric AKT inhibitor suitable for clinical development

Abstract

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.

Figure 1

Early development of novel allosteric AKT inhibitors based on a bicyclic pyrido-oxazinone core. (A) Chemical structures of early lead chemotypes used as starting points for medicinal chemistry development. (B) Hit and lead-optimization strategies employed. (C) Chemical structure of development candidate ALM301.

Figure 2

Biochemical inhibition of AKT isoforms versus pH domain null isoforms and ALM301 docking studies. (A) Biochemical inhibition of AKT1/AKT2/AKT3 and (B) related PH domain null isoforms using ALM301. (C) Docked pose of ALM301 in the allosteric pocket of AKT2 based on the crystal structure data generated using the closely related analogue compound 3. (D) Interaction diagram based on the docking of ALM301 with the allosteric pocket of AKT2 depicting key amino acid interactions.

Figure 3

Cellular profiling and validation of development candidate ALM301 in MCF-7 cells both as a single agent and in combination with tamoxifen. (A) Cell line profiling of ALM301 and MK-2206 in a panel of 24 cell lines including normal and tumour cells. (Black = cancer cell lines, White = normal cell lines). (B) Inhibition of MCF-7 cellular proliferation with 5 µM of ALM301 over 7 days using live cell imaging with an Incucyte imaging system. (C) Western blotting demonstrating inhibition of pAKT and pGSK3β in MCF-7 cells at various concentrations and timepoints up to 48 h. (D) Clonogenic cell survival of MCF-7 cells treated with ALM301 over a concentration range of 0.001–10 µM (IC₅₀ = 100 nM in this assay). (E) Cell cycle flow cytometry of MCF-7 cells treated with concentration range of 0.1–100 µM of ALM301 for 72 h. (F) Inhibition of MCF-7 cell viability using a combination of ALM301 and Tamoxifen at concentrations of 10 nM and 1 µM respectively. (G) Cell cycle flow cytometry of MCF-7 cells treated with ALM301 in combination with tamoxifen indicating apoptotic subG0 cell populations. Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Cellular profiling and validation of development candidate ALM301 in MCF-7 cells both as a single agent and in combination with tamoxifen. (A) Cell line profiling of ALM301 and MK-2206 in a panel of 24 cell lines including normal and tumour cells. (Black = cancer cell lines, White = normal cell lines). (B) Inhibition of MCF-7 cellular proliferation with 5 µM of ALM301 over 7 days using live cell imaging with an Incucyte imaging system. (C) Western blotting demonstrating inhibition of pAKT and pGSK3β in MCF-7 cells at various concentrations and timepoints up to 48 h. (D) Clonogenic cell survival of MCF-7 cells treated with ALM301 over a concentration range of 0.001–10 µM (IC₅₀ = 100 nM in this assay). (E) Cell cycle flow cytometry of MCF-7 cells treated with concentration range of 0.1–100 µM of ALM301 for 72 h. (F) Inhibition of MCF-7 cell viability using a combination of ALM301 and Tamoxifen at concentrations of 10 nM and 1 µM respectively. (G) Cell cycle flow cytometry of MCF-7 cells treated with ALM301 in combination with tamoxifen indicating apoptotic subG0 cell populations. Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

PK/PD and efficacy studies using development candidate ALM301 in A549 and MCF-7 xenograft models. (A) PK/PD relationship for ALM301 in A549 lung cancer xenograft model analysing levels of pAKT^S473 in tumours using western blotting over 24 h after a single oral dose of either ALM301 (10, 30 or 100 mg/kg) or MK-2206 (100 mg/kg). (B) Representative immunohistochemistry sections of phospho AKT stained tumour samples (brown) treated with a single dose of either vehicle only or 30 mg/kg of ALM301 over 24 h period (C) Growth inhibition curves in an A-549 tumour xenograft model treated with either vehicle (p.o., q.d. × 28, n = 10/group) or ALM301 (p.o. 10/30 mg/kg q.d. × 28 or p.o. 100 mg/kg q.d × 3 then q.o.d. × 25, n = 10/group). Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001. (D) Growth inhibition curves of MCF-7 tumours treated with either vehicle (p.o., q.d. × 49, n = 10/group), ALM301 (p.o. 3 or 10 mg/kg q.d. × 49 n = 10/group), tamoxifen (p.o. 5 mg/kg q.d. × 49, n = 10/group) or ALM301 (p.o. 3 or 10 mg/kg q.d. × 49 n = 10/group) in combination with tamoxifen (p.o. 5 mg/kg q.d. × 49). Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001. ADME: Absorption, distribution, metabolism, excretion; DMPK: drug metabolism and pharmacokinetics; PK/PD: pharmacokinetics and pharmacodynamics.

Figure 4

PK/PD and efficacy studies using development candidate ALM301 in A549 and MCF-7 xenograft models. (A) PK/PD relationship for ALM301 in A549 lung cancer xenograft model analysing levels of pAKT^S473 in tumours using western blotting over 24 h after a single oral dose of either ALM301 (10, 30 or 100 mg/kg) or MK-2206 (100 mg/kg). (B) Representative immunohistochemistry sections of phospho AKT stained tumour samples (brown) treated with a single dose of either vehicle only or 30 mg/kg of ALM301 over 24 h period (C) Growth inhibition curves in an A-549 tumour xenograft model treated with either vehicle (p.o., q.d. × 28, n = 10/group) or ALM301 (p.o. 10/30 mg/kg q.d. × 28 or p.o. 100 mg/kg q.d × 3 then q.o.d. × 25, n = 10/group). Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001. (D) Growth inhibition curves of MCF-7 tumours treated with either vehicle (p.o., q.d. × 49, n = 10/group), ALM301 (p.o. 3 or 10 mg/kg q.d. × 49 n = 10/group), tamoxifen (p.o. 5 mg/kg q.d. × 49, n = 10/group) or ALM301 (p.o. 3 or 10 mg/kg q.d. × 49 n = 10/group) in combination with tamoxifen (p.o. 5 mg/kg q.d. × 49). Paired t-test was used to derive p values (n = 10/group), *p < 0.05, **p < 0.01, ***p < 0.001. ADME: Absorption, distribution, metabolism, excretion; DMPK: drug metabolism and pharmacokinetics; PK/PD: pharmacokinetics and pharmacodynamics.