Inhibition of TRAF6-Ubc13 interaction in NFkB inflammatory pathway by analyzing the hotspot amino acid residues and protein–protein interactions using molecular docking simulations

Highlights

• TRAF6-Ubc13 interactions. PDTC- anti-inflammatory drug. New proposed drug candidate. TRAF6-drug interactions.

Abstract

Protein–protein interactions (PPIs) are important in most of the biochemical processes. Hotspot amino acid residues in proteins are the most important contributors for proper protein–protein interactions. Hotspot amino acid residues have been looked down upon as important therapeutic targets in inhibiting PPIs. Interaction between TRAF6 and Ubc13 is a crucial point in the NFkB inflammatory pathway. Dysfunction of the NFkB pathway is associated with numerous human diseases including cancer and neurodenegeration disorders. Ubc13 also interacts specifically to TRAF6 and not with other proteins of the TRAF family and this makes the TRAF6-Ubc13 complex an important target for specific inhibition. Hence, interfering with the TRAF6-Ubc13 association may prove effective in suppressing the NFkB disease pathway. In the present study, we searched the TRAF6-Ubc13 interaction interface to analyze their binding hotspot amino acid residues using various computational techniques. Heterocyclic compounds are known for their medicinal properties. We screened for heterocyclic analogues to the known TRAF6 inhibitor PDTC, to predict a better inhibitor using in silico protein–ligand and protein–protein interaction studies. Our in silico prediction results suggest that tetrahydro-2-thiophenecarbothioamide (Chemspider ID 36027528) binds one of the major hot-spot residues of TRAF6-Ubc13 interface and can be a better alternative in suppressing TRA6-Ubc13 complex formation in chronic inflammation than PDTC.

Introduction

Proteins govern various cellular processes by interacting with each other through their interfaces. Dysfunctional PPIs often result in the manifestation of disease conditions including cancer (Ali and Bagchi, 2015, Garner and Janda, 2011). This property makes PPIs an attractive therapeutic target. PPI interface area is generally large, less conserved, flat (∼1000–2000 Å²) and less shallow (Hwang et al., 2010) which makes it difficult to be targeted by small molecules which possess a comparatively smaller area (∼300–500 Å²) (Fuller et al., 2009). This makes hampering or disrupting PPI by small molecules quite a challenge. Several studies suggest that small molecules targeting the “binding hot-spot” amino acid residues rather than the whole PPI interface might prove to be very effective as drugs (Arkin and Wells, 2004, Cukuroglu et al., 2014). All residues in the PPI interfaces do-not have the same energetic contributions towards binding interactions; some amino acid residues are the major contributors to PPIs. These amino acid residues are termed as “hot-spot” residues. These “hot-spot” residues also play important parts in conferring protein stability (Guo et al., 2014). Hot-spot residues are characterized computationally by the method of alanine scanning mutagenesis experiment in which each interacting residue is mutated to alanine, getting rid of side chain atoms beyond C_β and then calculating the overall change in binding free energy (ΔΔG). Change in binding free energy is defined as ΔG^mut − ΔG^wt, where ΔG^mut and ΔG^wt are the binding free energies of alanine substituted and wild type amino acids in the protein complexes respectively (Clackson and Wells, 1995, Kenneth Morrow and Zhang, 2012). By definition, hot-spot residues are the ones whose substitution by alanine leads to a decrease of binding free energy (ΔΔG) > 2 kcal/mol. Density of hot-spot residues characteristically swathe 10% of binding site residues. Hot-spot residues are not randomly scattered across the PPI interfaces but rather are tightly packed together to form hot-regions (Bogan and Thorn, 1998).

The protein TRAF6 belongs to the family of tumor necrosis factor receptor-associated factor (TRAF) and is an E3 ligase enzyme. It plays a key role in IL-1 mediated NFkB pro-inflammatory pathway (Twomey et al., 2009). NFkB is a prime mediator of the cellular responses to environmental changes. It is known to be involved in development of neurons, proliferation of cells, cell death and immunological responses to infection and inflammation. Dysfunction or dysregulation of this pathway is associated with diseases like chronic inflammation (Lawrence, 2009), immunodeficiency (Smahi et al., 2002) and various human malignancies (Karin, 2009). Neurodegenerative diseases like Alzeimers (Granic et al., 2009) and Parkinsons (Panet et al., 2001) are also known to be associated with NFkB pathway. The structural characteristic of TRAF6 includes an N-terminal RING (really interesting new gene) domain preceded by four Zn-finger domains and a C-terminal coiled coil (CC) and TRAF-C domains. The RING domain of TRAF6 is known to coordinate with two Zn²⁺ ions into its active site. The RING domain physically interacts with the protein Ubc13 which is an E2 enzyme to carry-out the process of ubiquitination of IkB (Wu and Arron, 2003, Lamothe et al., 2008). Hence, inhibition of E2 binding ability of RING TRAF6 can suppress NFkB inflammation pathway by hampering the degradation of IkB by ubiquitination. Pyrrolidine dithiocarbamate (PDTC), a stable pyrroline derivative of dithiocarbamate, is known to lower NFkB activity by inhibiting E3 ubiquitin ligase activity (Schreck et al., 1992, Ziegler-Heitbrock et al., 1993). Heterocyclic compounds have long been known for their potential therapeutic applications in medicinal chemistry. By definition heterocyclic compounds are cyclic compounds having at least one element other than carbon(s) in the ring (Martins et al., 2015).

In this present study, we used molecular docking studies to screen for heterocyclic analogues of PDTC and test their potential for predictive binding of the hotspot residues in TRAF6-Ubc13 interaction interface. Ligands were further selected on the basis of interaction and binding free energies and making strong association (H-bond) with the hot-spot residues. We further analyzed the ligand bound complex of TRAF6 for its specific binding ability to Ubc13. Our study may therefore be useful in future drug development endeavors for synthesis of new drugs to target the NFkB inflammation pathway.