In Silico Studies on GCP-Lys-OMe as a Potential 14-3-3σ Homodimer Stabilizer

Abstract

14-3-3 sigma is a vital negative cell cycle regulator. Its expression is consistently downregulated in many types of cancer through gene promoter hypermethylation or proteasomal degradation. 14-3-3 sigma needs to form a homodimer to be functional, while dimers are less prone to degradation than monomers. This suggests that a homodimer stabilizer may increase the tumor suppressive activities of 14-3-3 sigma. However, no known homodimer stabilizer of 14-3-3 sigma has been reported to date. Therefore, this study attempts to test the potential capability of GCP-Lys-OMe (previously reported to bind at the dimer interface of 14-3-3 zeta isoform), to bind and stabilize the 14-3-3 sigma homodimer. In silico docking of GCP-Lys-OMe on 14-3-3 sigma showed more favorable interaction energy (−9.63 kcal/mole) to the dimer interface than 14-3-3 zeta (−7.73 kcal/mole). Subsequent 100 ns molecular dynamics simulation of the GCP-Lys-OMe/14-3-3 sigma complex revealed a highly stable interaction with an average root-mean-square deviation of 0.39 nm (protein backbone) and 0.77 nm (ligand atoms). More contacts between residues at the homodimer interface and a smaller coverage of conformational space of protein atoms were detected for the bound form than for the apo form. These results suggest that GCP-Lys-OMe is a potential homodimer stabilizer of 14-3-3 sigma.

In Silico Prediction and Biophysical Validation of Novel 14-3-3σ Homodimer Stabilizers

“…A multiparameter (size and count rate) analysis fitting algorithm was used for calculating the aggregation temperature, T agg . For DLS analysis on 14-3-3σ proteins at selected temperatures (25,42,50, and 60 °C), the volume distribution analysis of the protein across a range of different temperatures was conducted using a Litesizer100 (Anton Paar GmbH, Austria), with three measurements recorded at each temperature.…”

“…Our previous in silico work on GCP-Lys-OMe revealed that the basic functional groups of GCP-Lys-OMe, i.e., the guanidino and the primary amine groups, separated by approximately 12 Å, are critical for its interaction with the acidic residues at the dimer interface of 14-3-3σ, i.e., Glu20A, Glu20B, Glu91A, and Glu91B. 50 As the guanidino group is also observed in the side chain of Arg, while the primary amine group is observed at the N-terminus as well as the side chain of Lys, we hypothesized that a free N-terminus dipeptide with either one or both types of residues, with the two basic functional groups separated by a similar distance as observed in GCP-Lys-OMe, may also bind to the dimer interface of 14-3-3σ. Therefore, four different categories of dipeptides, i.e., Lys− Arg (KR), Arg−Lys (RK), Arg−Arg (RR), and Lys−Lys (KK), with different combinations of stereoisomers (LL, DL, DD, and LD) (to cater for the different distances between the two basic functional groups in the peptides) were generated (Table 1).…”

“…Previously, we have shown using a FTMap web server 54 that the amphipathic binding groove in each 14-3-3σ monomer and the cavity at the dimer interface of the 14-3-3σ are the two potential druggable sites of 14-3-3σ. 50 However, to date, only ligands at the amphipathic pocket of 14-3-3σ are known. To rule out the possibility that peptide 3 binds to the amphipathic groove of 14-3-3σ, a competition [ 1 H]-CPMG NMR assay with a known binder of 14-3-3σ at the amphipathic groove, i.e., the ExoS peptide ( 420 QGLLDALDLAS 430 ), 55 was conducted.…”

“…49 Recently, we have used in silico molecular docking and molecular dynamics (MD) simulation techniques to investigate the possibility of GCP-Lys-OMe to stabilize the 14-3-3σ homodimer. 50 Our results revealed that GCP-Lys-OMe was not only able to target the dimer interface of 14-3-3σ but also increased the contacts between the monomers. These results encourage us to study if GCP-Lys-OMe or its peptide analogues are also able to stabilize the 14-3-3σ homodimer in vitro.…”

“…Thus, a virtual library of 16 peptide analogues of GCP-Lys-OMe was proposed based on the key functional groups observed for GCP-Lys-OMe binding to 14-3-3σ in our previous study. 50 In silico molecular docking of all peptide analogues was performed and their interacting residues and binding free energies were compared to that of GCP-Lys-OMe. Subsequently, 5 replicas of 100 ns MD simulations each were conducted on both the complex between 14-3-3σ and the highest ranked peptide as well as the 14-3-3σ protein alone, and the differences between the two systems were statistically analyzed using the paired t-test to predict the ability of the peptide to stabilize the 14-3-3σ homodimer.…”