Interaction of the σ₂ Receptor Ligand PB28 with the Human Nucleosome: Computational and Experimental Probes of Interaction with the H2A/H2B Dimer

Abstract

Sigma-2 (sigma(2)) binding sites are an emerging target for anti-neoplastic agents due to the strong apoptotic effect exhibited by sigma(2) agonists in vitro and the overexpression of these sites in tumor cells. Nonetheless, no sigma(2) receptor protein has been identified. Affinity chromatography using the high-affinity sigma(2) ligand PB28 and human SK-N-SH neuroblastoma cells was previously utilized to identify sigma(2) ligand binding proteins, specifically histones H1, H2A, H2B, and H3.3a. To rationalize this finding, homology modeling and automated docking studies were employed to probe intermolecular interactions between PB28 and human nucleosomal proteins. These studies predicted interaction of PB28 with the H2A/H2B dimer at a series of sites previously found to be implicated in chromatin compaction and nucleosomal assembly. To experimentally verify this prediction, a competitive binding assay was performed on the reconstituted H2A/H2B dimer using [(3)H]PB28 as radioligand, and an IC(50) value of 0.50 nM was determined for PB28 binding. In addition, [(3)H]PB28 was found to accumulate with up to a fivefold excess in nuclear fractions over cytosolic fractions of SK-N-SH and MCF7 cells, indicating that PB28 is capable of entering the nucleus to interact with histone proteins. In conjunction with computational results, these data suggest that PB28 may exert its cytotoxic effect through direct interaction with nuclear material.

Elements in support of the ‘non-identity’ of the PGRMC1 protein with the σ2 receptor

“…Colabufo et al (2006) suggested that σ 2 receptor could be related to histone proteins (Abate et al, 2010), whereas recent studies proposed σ 2 binding site located in the progesterone receptor membrane component 1 (PGRMC1) (Xu et al, 2011). Although in a few recent papers the identity of the σ 2 receptor with PGRMC1 appears as accepted Mir et al, 2012Mir et al, , 2013 there is still controversy about that (Ruoho et al, 2013;van Waarde et al, 2014).…”

1‐Cyclohexyl‐4‐(4‐arylcyclohexyl)piperazines: Mixed σ and Human Δ8–Δ7 Sterol Isomerase Ligands with Antiproliferative and P‐Glycoprotein Inhibitory Activity

“…[a] Dr. C. with (R,S)-1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1, (R,S)-PB28; Figure 1) generated the hypothesis that the s 2 receptor could be a histone protein, [21,22] while other s 2 receptor ligands demonstrated that this subtype is not localized within the nucleus. [23] Despite these contradictory results, existing evidence shows that s 1 antagonists and s 2 agonists exert antiproliferative activity in several tumor cell lines.…”

Classes of Sigma2 (σ2) Receptor Ligands: Structure Affinity Relationship (SAfiR) Studies and Antiproliferative Activity

“…Therefore, the authors hypothesized that 2 receptors may be histones, or histone binding proteins or that PB28 binds histones as well as at 2 receptors. A homology modeling study rationalized PB28 binding to histone proteins and in particular to the H2A-H2B histone dimer, providing a possible explanation for the antiproliferative action of PB28 [56]. However, other findings from 2 receptor fluorescent ligands localize 2 subtypes in several organelles except the nucleus [57][58][59] making the histone hypothesis weaker.…”