Dual Binding to Orthosteric and Allosteric Sites Enhances the Anticancer Activity of a TRAP1-Targeting Drug

Abstract

The molecular chaperone TRAP1 is the mitochondrial paralog of Hsp90 and is overexpressed in many cancer cells. The orthosteric ATP-binding site of TRAP1 has been considered the primary inhibitor binding location, but TRAP1 allosteric modulators have not yet been investigated. Here, we generated and characterized the Hsp90 inhibitor PU-H71, conjugated to the mitochondrial delivery vehicle triphenylphosphonium (TPP) with a C10 carbon spacer, named SMTIN-C10, to enable dual binding to orthosteric and allosteric sites. In addition to tight binding with the ATP-binding site through the PU-H71 moiety, SMTIN-C10 interacts with the E115 residue in the N-terminal domain through the TPP moiety and subsequently induces structural transition of TRAP1 to a tightly packed closed form. The data indicate the existence of a druggable allosteric site neighboring the orthosteric ATP pocket that can be exploited to develop potent TRAP1 modulators.

TRAP1 Chaperones the Metabolic Switch in Cancer

“…Complex II/SDH is an iron–sulfur cluster-containing protein complex that functions to transfer electrons from succinate to coenzyme Q10-ubiquinone (Complex III) [ 48 ]. In agreement with the understanding of Hsp90 function, TRAP1 maintains SDH in a partially unfolded state [ 49 ], and TRAP1 inhibition releases active SDH, leading to an increase in its activity [ 27 , 44 , 50 , 51 , 52 ]. Further, SDH activity [ 44 , 53 , 54 ] and the oxygen consumption rate [ 6 , 55 ] are inversely correlated with TRAP1 expression, implicating TRAP1 in promoting the Warburg effect [ 56 ].…”

“…When the TPP was modified to have a 10-length carbon chain (as opposed to the standard 6-length carbon chain), this so-called SMTIN-C10 induced structural changes to TRAP1 and demonstrated increased inhibition of TRAP1 [ 52 ]. SMTIN-C10 was found to bind to an allosteric binding site at E115 in the N-terminal domain of TRAP1, in addition to binding to the ATP pocket, resulting in TRAP1 adopting a closed formation [ 52 ]. This long linker approach was adapted for other TRAP1 inhibitors as well, including Mitoquinone.…”

Mitoquinone Inactivates Mitochondrial Chaperone TRAP1 by Blocking the Client Binding Site

“…(C) Binding affinity of alkyl TPPs to the ATP binding pocket of TRAP1. hTRAP1 was incubated with alkyl TPPs in the presence of PU-H71-FITC3 and analyzed by fluorescence polarization. mP, millipolarization.…”

“…This approach successfully converted cytoplasmic Hsp90 inhibitors (moderate activity) to mitochondria-accumulating TRAP1 inhibitors (potent cytotoxic activity). Examples include gamitrinib, SMTIN-P01, and SMTIN-C10. ,− …”

Allosteric Regulation at the Crossroads of New Technologies: Multiscale Modeling, Networks, and Machine Learning

“…The computational predictions were combined with experimental validation showing that the selected molecules bind the allosteric sites of Hsp90, exhibit anti-proliferative activity in different tumor cell lines, and destabilize Hsp90 client proteins (Morra et al, 2010 ). The recent series of studies by Colombo and colleague have reported results of computer-aided design and synthesis of new allosteric ligands with micromolar to nanomolar anticancer activities, demonstrating the power of computational approaches in discovering allosteric modulators that can probe the relationships between structure dynamics and function of the Hsp90 proteins and regulatory complexes with client proteins (Sattin et al, 2015 ; D'Annessa et al, 2017 ; Masgras et al, 2017 ; Ferraro et al, 2019 ; Hu et al, 2020 ; Sanchez-Martin et al, 2020 ). Computational targeting of the Hsp90 client proteins based on the prediction of locally unstable substructures in proteins was used to develop potent probes and peptides blocking Hsp90-client interactions (Colombo et al, 2020 ).…”