Journal: ACS Central Science

Article Title: Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization

doi: 10.1021/acscentsci.2c00013

Figure Lengend Snippet: Rational design and synthesis of tripyrimidonamides. (a) Cryo-EM structure of the dimer of human Hsp90β (PDB ID 5FWK), shown in surface and cartoon representations. For one of the Hsp90 monomers, the N-terminal domain (NTD) is colored in red, the middle domain in beige, and the C-terminal domain (CTD) in blue. Above and below the protein structure, the structures of Hsp90i and their potential binding sites (see refs ( − ), color-coded according to the domains) are shown. (b) Dimeric CTD of human Hsp90β with the two monomers in blue and white. Helices H4, H4′, H5, and H5′ of the CTDs form the dimerization interface. (c) Residues forming the CTD dimerization interface in human Hsp90α are primarily located on helices H4, H4′, H5, and H5′. (d) Tripyrimidones can adopt conformations resembling the side chain orientation of an α-helix in i , i + 4, and i + 7 positions. (e) Synthesis of tripyrimidonamides: (a) COMU, DMF, r.t., 18 h; (b) NaOH, MeOH, 80 °C, 6 h; (a) 2a – 2d , COMU, DMF, r.t., 18 h; (c) 6 via 5b , BBr 3 , DCM, −78 °C, 1 h, r.t., 1 h; (d) via 7a via 5c and 7b via 5d , H 2 , Pd(C), MeOH, DCM, r.t., 1 h.

Article Snippet: An evaluation of the binding affinity of compounds toward the ATP pocket of Hsp90 NTD was determined by a competitive binding assay against FITC-labeled geldanamycin (GM) using the Hsp90 NTD assay kit (50293, BPS Bioscience).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: ACS Central Science

Article Title: Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization

doi: 10.1021/acscentsci.2c00013

Figure Lengend Snippet: Selection of 5b as a lead candidate. (a) Schematic view of the Hsp90 dimerization assay using Autodisplay. (b) Flow cytometry measurements of the inhibition of dimerized Hsp90α displayed on E. coli cells. E. coli BL21 (DE3) cells displaying Hsp90α incubated with 1 μM FITC-labeled p53 lead to a high cellular fluorescence indicating dimerization of Hsp90α. The value obtained was set as 0% inhibition. In contrast, E. coli cells without displaying Hsp90α (control cells) show no cellular fluorescence. The value obtained here was set as 100% inhibition. Preincubation of E. coli cells with surface-displayed Hsp90α with 50 μM of the respective substance leads to a lowered cellular fluorescence intensity indicating a lowered binding affinity of FITC-labeled p53 to surface-displayed Hsp90α. These values were set in relation to obtain the relative inhibition of dimerization. (c) Apparent K D values of the purified CTD of Hsp90α and the respective substance measured via the MST method. A constant amount of 50 nM labeled CTD of Hsp90 was used, and three independent measurements were performed. The resulting mean values were determined and used in the K D fit formula. (d) Cellular viability assessment of a leukemic cell line (K562) measured by incubating with the indicated inhibitors for 72 h, followed by a viability measurement using an ATP-based Celltiter Glo assay. (e) Selection of 5b as a lead candidate on the basis of high inhibition of Hsp90α dimerization, low apparent K D , and low IC 50 (μM) in a tested leukemic cell line.

Article Snippet: An evaluation of the binding affinity of compounds toward the ATP pocket of Hsp90 NTD was determined by a competitive binding assay against FITC-labeled geldanamycin (GM) using the Hsp90 NTD assay kit (50293, BPS Bioscience).

Techniques: Selection, Flow Cytometry, Inhibition, Incubation, Labeling, Fluorescence, Binding Assay, Purification, Glo Assay

Journal: ACS Central Science

Article Title: Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization

doi: 10.1021/acscentsci.2c00013

Figure Lengend Snippet: Specificity of 5b against Hsp90 CTD and its cochaperone function. (a) Recombinant (full-length) Hsp90α (1 μg) was incubated with 5b at indicated concentrations, followed by digestion with thermolysin. Treated protein samples were electrophoresed (SDS-PAGE) and immunoblotted with anti-Hsp90α for detecting the protection of Hsp90α protein by 5b (the upper band is protected from proteolysis). (b) A cell-free thermal shift assay was performed by incubating recombinant Hsp90α CTD protein with 5b at an increasing temperature (up to 95 °C). The melting temperature ( T m ) without inhibitors (DMSO) was used as a control. (c) Dose-dependent intracellular (K562 cells) thermal stabilization (CETSA ITDRF ) of Hsp90 after 5b incubation (24 h) at its increasing concentration (1.25–5 μM). (d) 5b inhibits the Hsp90α chaperone function, comparable to TM and GM, in the cell-free luciferase refolding assay, where the incubation of the inhibitors prevented the rabbit reticulocyte lysate (a source of Hsp90)-assisted refolding of denatured luciferase. (e) Incubation of 5b blocked the binding of Hsp90 CTD-interacting cochaperone (PPID) in TR-FRET measurements. (f) 5b did not reduce the amount of Hsp90-bound FITC-labeled GM and, therefore, does not compete for the GM binding pocket of full-length Hsp90α. Unlabeled GM, GP, PUH71, and TM served as positive controls and NB and CA1 as negative controls.

Article Snippet: An evaluation of the binding affinity of compounds toward the ATP pocket of Hsp90 NTD was determined by a competitive binding assay against FITC-labeled geldanamycin (GM) using the Hsp90 NTD assay kit (50293, BPS Bioscience).

Techniques: Recombinant, Incubation, SDS Page, Thermal Shift Assay, Concentration Assay, Luciferase, Binding Assay, Labeling

Journal: ACS Central Science

Article Title: Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization

doi: 10.1021/acscentsci.2c00013

Figure Lengend Snippet: Effect of 5b on Hsp90 oligomeric species and CTD-mediated dimerization. (a) Recombinant Hsp90α CTD was incubated with 63 μM BS 3 cross-linker with (at the indicated concentration) or without 5b , followed by immunoblotting with the anti-Hsp90 (AC88) antibody. (b) The scattering data of Hsp90α CTD is shown in black dots, with gray error bars. The ab initio DAMMIF model fit is shown as a red line. The intensity is displayed as a function of momentum transfer s . (c) The volumetric envelope, calculated from the scattering data using DAMMIF, is shown as a blue surface. The monomers of the predicted Hsp90 CTD dimer model are shown in green and cyan. Superimposing was performed using SUPCOMB. (d) The radius of gyration ( R g ) of the different Hsp90α CTD protein samples was calculated using the Guinier approximation. The theoretical R g of the tetramer was calculated using CRYSOL based on the structure PDB ID 3q6m. (e) Native Hsp90 complexes in K-562 (24 h administration of 5b ) were identified by running blue native (BN) gels followed by immunoblotting analysis. The cytotoxic concentration of 5b resulted in the potent disruption of Hsp90α, Hsp90β, Hsp40, and Hsp27 complexes and monomers/dimers. AUY922 exposure elevated the expression of HSR associated protein complexes and monomers/dimers (Hsp40 and Hsp27), whereas Hsp60 served as loading controls.

Article Snippet: An evaluation of the binding affinity of compounds toward the ATP pocket of Hsp90 NTD was determined by a competitive binding assay against FITC-labeled geldanamycin (GM) using the Hsp90 NTD assay kit (50293, BPS Bioscience).

Techniques: Recombinant, Incubation, Concentration Assay, Western Blot, Expressing