An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine‐56

Abstract

The stress‐inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine‐targeted irreversible inhibitor. Using rational design, we adapted a validated 8‐N‐benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine‐56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.

Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition

“…13 Nonetheless, interest in this nucleophile is rapidly gaining traction in the scientific community. [14][15][16][17][18] The heterodimeric lipid kinase phosphoinositide 3kinase delta (PI3Kδ) 19,20 has been targeted specifically over the related PI3Kα, β and γ isoforms for the treatment of a variety of diseases. 21,22 A number of selective reversible PI3Kδ small molecule inhibitors have entered clinical trials, with Zydelig recently obtaining FDA approval as a second-line treatment for relapsed follicular B-cell non-Hodgkin lymphoma, and relapsed chronic lymphocytic leukemia.…”

Lysine‐Targeting Covalent Inhibitors

“…[75] These proteins have become popular targets in oncology but have proven particularly difficult to inhibit owing to the high affinity of their endogenous substrates.I na na ttempt to develop cell-active chemical probes to study HSP70, we used rational design to modify the validated 8-N-benzyl adenosine reversible inhibitor 33 [76] with an electrophilic aliphatic acrylate,i nitially targeting ap roximal active site cysteine(Cys17;F igure 7E). [77] 11. [77] 11.…”

“…Further analysis confirmed specific covalent bond formation and irreversible inhibition, but studies utilizing single-point mutations unambiguously established that 34 was actually binding to the highly conserved but non-catalytic active-site Lys56 (Figure 7F). [77]…”

Auf Lysin zielende, kovalente Inhibitoren

“…Wir wollten zellaktive chemische Sonden zur Untersuchung von HSP70 entwickeln und nutzten einen rationalen Design‐Ansatz, um den validierten reversiblen 8‐ N ‐Benzyladenosin‐Inhibitor 33 mit einem elektrophilen aliphatischen Acrylat so zu modifizieren, dass er zunächst auf ein Cystein in der Nähe des Wirkorts zielte (Cys17; Abbildung E). Die weitere Analyse bestätigte zwar die Bildung einer spezifischen kovalenten Bindung und die irreversible Inhibierung, doch Untersuchungen mit Einzelpunktmutationen ergaben zweifelsfrei, dass 34 in Wirklichkeit an das hoch konservierte, aber nichtkatalytische Lys56 des Wirkorts gebunden hatte (Abbildung F) …”