Degradation of Akt using protein-catalyzed capture agents

Abstract

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets.

Keywords: Akt; PROTAC; click chemistry; protein-catalyzed capture agents.

Figure 1. Akt activating PCC, tri_a, and exploiting the modularity of this PCC

(A) Structure of Akt-activating N-terminal triligand, tri_a. The lysine residue between the PEG spacers can be further functionalized with fluorescein or the degradation-inducing Hif-1α peptide. Complete structures are shown in Figure S1. R = H is referred to as CPP-tri_a, R = fluorescein is CPP-tri_a-FL, and R = ALAPYIP (Hif-1α degradation peptide) is CPP-tri_a-PR. (B) Live-cell confocal images of the fluorescein-labeled capture agent CPP-tri_a-FL delivered into U87 cells.

Figure 2. tri_a activates Akt in cells

(A) Western blots of SKOV3 cells treated with 50 μM tri_a Akt-activating capture agent. Densitometry was performed on the p-GSK-3β blot and the relative intensities were plotted on the bar graph below the blotting assays. (B) XTT cell proliferation assay results from OVCAR3 and SKOV3 cells treated with 50 μM tri_a for times varying from 1-5 days. A student’s t test was used to determine significant differences where * = p-value ≤ 0.05, and *** = p-value ≤ 0.01. See Figure S2 for parallel inhibition experiments.

Figure 3. Tri_a-PROTAC knocks down Akt in cells

(A) In-cell ELISA measurement of Akt in OVCAR3 cells treated with tri_a-PROTAC containing the degradation-inducing Hif-1α sequence for various times. (B) In-cell ELISA measurement of Akt after 4 hour treatment at variable tri_a-PROTAC doses. Results show dose-dependent degradation of Akt with an EC₅₀ value of 128±19 μM. The data are normalized to the untreated control and relative levels of Akt/cell are plotted. Figure S3 contains additional degradation data with CPP-tri_1p-PR.