In silico identification and characterization of small-molecule inhibitors specific to RhoG/Rac1 signaling pathway

Abstract

Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. RhoG is one of the less explored Rho GTPases with significant sequential and structural homology with Rac1. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), maybe an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Z-score of –6.46, and 87.96% of residues had an averaged 3D–1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our results from docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1 based inhibitor, EHop-016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.

Communicated by Ramaswamy H. Sarma

Keywords:

• Rho GTPases
• guanine nucleotide exchange factors (GEFs)
• GTPase activating proteins (GAPs)
• molecular docking
• molecular dynamic simulation
• MM/PBSA
• homology modelling

Acknowledgment

The authors acknowledge the Institute Computer Centre (ICC), IIT Roorkee, to provide the required computing facilities, and Prof. Jagnehswar Dandpat, and Mr. Atala Bihari Jena from the Department of Biotechnology, Utkal University for their valuable discussions and technical inputs.

Disclosure statement

The authors declare no potential conflicts of interest.

Additional information

Funding

We are thankful to the Department of Biotechnology, Govt. of India (No. BT/010/IYBA/2017/04) and Science Engineering Research Board, Govt. of India (CRG-2020-0025) to P.P.S. and ICMR fellowship to PD.