Potential of Withaferin-A, Withanone and Caffeic Acid Phenethyl ester as ATP-competitive inhibitors of BRAF: A bioinformatics study

Abstract

Serine/threonine-protein kinase B-raf (BRAF) plays a significant role in regulating cell division and proliferation through MAPK/ERK pathway. The constitutive expression of wild-type BRAF (BRAF^WT) and its mutant forms, especially V600E (BRAF^V600E), has been linked to multiple cancers. Various synthetic drugs have been approved and are in clinical trials, but most of them are reported to become ineffective within a short duration. Therefore, combinational therapy involving multiple drugs are often recruited for cancer treatment. However, they lead to toxicity and adverse side effects. In this computational study, we have investigated three natural compounds, namely Withaferin-A (Wi-A), Withanone (Wi-N) and Caffeic Acid Phenethyl ester (CAPE) for anti-BRAF^WT and anti-BRAF^V600E activity. We found that these compounds could bind stably at ATP-binding site in both BRAF^WT and BRAF^V600E proteins. In-depth analysis revealed that these compounds maintained the active conformation of wild-type BRAF protein by inducing αC-helix-In, DFG-In, extended activation segment and well-aligned R-spine residues similar to already known drugs Vemurafenib (VEM), BGB283 and Ponatinib. In terms of binding energy, among the natural compounds, CAPE showed better affinity towards both wild-type and V600E mutant proteins than the other two compounds. These data suggested that CAPE, Wi-A and Wi-N have potential to block constitutive autophosphorylation of BRAF and hence warrant in vitro and in vivo experimental validation.

Keywords: ATP-Competitive inhibitors and cancer; BRAF; BRAF V600E mutant; Caffeic acid phenethyl ester; Withaferin-A; Withanone.

Graphical abstract

Fig. 1

(A) Structure of compounds selected for the study. (B) Structure of BRAF-MEK1 dimer (PDB ID: 4MNE) selected for the study. (C) Structure of BRAF^WT-BGB283 dimer complex. The structural elements around ATP/ligand-binding site of protein are highlighted that includes activation loop (shown in firebrick red color), αC-helix (orange) and P loop (salmon). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

The changes in the structure of the BRAF^WTand BRAF^V600Ewith and without ligands. (A) Superimposition of active BRAF^WT apo protein (grey) with BRAF^WT -ATP complex (yellow-red). (B) Superimposition of BRAF^V600E apo protein structure (grey) with that of BRAF^V600E-ATP complex (yellow-red). (C) Superimposition of active BRAF^WT -ATP complex (grey) with BRAF^WT -Ponatinib complex (yellow-red). (D) Superimposition of BRAF^V600E -ATP complex (grey) with that of BRAF^V600E-Ponatinib complex (yellow-red). (E) Superimposition of BRAF^WT protein structure (grey) with that of BRAF^WT-Ponatinib complex (yellow-red) at chain B. (F) Superimposition of BRAF^WT protein structure (grey) with that of BRAF^WT-Ponatinib complex (yellow-red) at chain C. (G) Superimposition of BRAF^V600E protein structure (grey) with that of BRAF^V600E-Ponatinib complex (yellow-red) at chain B. (H) Superimposition of BRAF^V600E protein structure (grey) with that of BRAF^V600E-Ponatinib complex (yellow-red) at chain C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Interaction of VEM and BGB283 at ATP-binding site of BRAF^WTand BRAF^V600Emutant. Superimposition of active BRAF^WT protein (grey) with BRAF^WT-BGB283 complex (yellow-red) at chain B (A) and chain C (B), BRAF^WT-VEM complex (yellow-red) at chain B (C) and chain C (D). Superimposition of BRAF^V600E protein structure (grey) with that of BRAF^V600E-BGB283 complex (yellow-red) at chain B (E) and chain C (F) and BRAF^V600E-VEM complex (yellow-red) at chain B (G) and chain C (H). The structural elements around ligand-binding site of BRAF^WT-inhibitor complex and BRAF^V600E-inhibitor complexes are highlighted that includes activation loop (shown in firebrick red color), αC-helix (orange), P loop (salmon) and R-spine residues (red lines representation). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Interaction of CAPE, Wi-A and Wi-N at ATP-binding site of BRAF^WTprotein. Superimposition of active BRAF^WT protein (grey) with BRAF^WT-Inhibitor complexes (yellow-red) including BRAF-CAPE complex at chain B (A) and chain C (B), BRAF-Wi-A complex at chain B (C) and chain C (D) and BRAF-Wi-N complex at chain B (E) and chain C (F). Structural elements of BRAF^WT-inhibitor complexes like activation segment (shown in firebrick color), αC-helix (orange), P loop (salmon) and R-spine residues (red lines representation). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Interaction of CAPE, Wi-A and Wi-N at ATP binding site of BRAF^V600Emutant protein. Superimposition of BRAF^V600E protein structure (grey) with that of BRAF^V600E mutant-Inhibitor complexes (yellow-red) including BRAF-CAPE complex at chain B (A) and chain C (B), BRAF-Wi-A complex at chain B (C) and chain C (D) and BRAF-Wi-N complex at chain B (E) and chain C (F). Structural elements of BRAF^V600E-inhibitor complexes like activation segment (shown in firebrick color), αC-helix (orange), P loop (salmon) and R-spine residues (red lines representation). Helical turn introduced in activation segment of BRAF^V600E-Wi-N complex is highlighted with cyan colored circle. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)