Design, synthesis, and evaluation of small molecule Hsp90 probes

Abstract

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.

Figure 1

Structures of Hsp90 inhibitors.

Figure 2

A) Interactions of Hsp90α (PDB ID: 2FWZ) with PU-H71 (ball and stick model) and compound 5 (tube model). B) Interactions of Hsp90α (PDB ID: 2VCI) with NVP-AUY922 (ball and stick model) and compound 10 (tube model). C) Interactions of Hsp90α (PDB ID: 3D0B) with compound 27 (ball and stick model) and compound 20 (tube model). Hydrogen bonds are shown as dotted yellow lines and important active site amino acid residues and water molecules are represented as sticks.

Figure 3

A) Hsp90 in K562 extracts (250 μg) was isolated by precipitation with PU-, SNX- and NVP-beads or Control-beads (80 μL). Control beads contain 2-methoxyethylamine, an Hsp90-inert molecule. Proteins in pull-downs were analyzed by Western blot. B) In MDA-MB-468 cell extracts (300 μg), PU-beads isolate Hsp90 in complex with its onco-client proteins, c-Kit and IGF-IR. To evaluate the effect of PU-H71 on the steady-state levels of Hsp90 onco-client proteins, cells were treated for 24 h with PU-H71 (5 μM). C) In K562 cell extracts, PU-beads (40 μL) isolate Hsp90 in complex with the Raf-1 and Bcr-Abl onco-proteins. Lysate = endogenous protein content; PU- and Control-beads indicate proteins isolated on the particular beads. The data are consistent with those obtained from multiple repeat experiments (n ≥ 2).

Figure 4

A) Hsp90-containing protein complexes from the brains of JNPL3 mice, an Alzheimer’s disease transgenic mouse model, isolated through chemical precipitation with beads containing a streptavidin-immobilized PU-H71-biotin construct or control streptavidin-immobilized D-biotin. Aberrant tau species are indicated by arrow. c1, c2 and s1, s2, cortical and subcortical brain homogenates, respectively, extracted from 6-month-old female JNPL3 mice (Right). Western blot analysis of brain lysate protein content (Left). B) Cell surface Hsp90 in MV4-11 leukemia cells as detected by PU-H71-biotin. The data are consistent with those obtained from multiple repeat experiments (n ≥ 2).

Scheme 1

Synthesis of PU-H71 beads (6). Reagents and conditions: (a) Cs₂C0₃, 1,3-dibromopropane, DMF, rt; (b) NH₂(CH₂)₆NHBoc (3), DMF, rt, 24h; (c) TFA, CH₂C1₂, rt; (d) Affigel-10, DIEA, DMAP, DMF.

Scheme 2

Synthesis of PU-H71-biotin (7). Reagents and conditions: (a) EZ-Link^® Amine-PE0₃-Biotin, DMF, rt.

Scheme 3

Synthesis of NVP-AUY922 beads (11). Reagents and conditions: (a) NH₂(CH₂)₆NHBoc (3), NaCNBH₃, AcOH, MeOH, rt; (b) BC1₃, CH₂C1₂, rt; (c) Affigel-10, DIEA, DMAP, DMF.

Scheme 4

Synthesis of SNX-2112 beads (21). Reagents and conditions: (a) p -toluene sulfonic acid, toluene, reflux, 1.5 h; (b) trifluoroacetic anhydride, Et₃N, THF, 55°C, 3 h, then methanol/NaOH rt, 3 h; (c) 2-bromo-4-fluorobenzonitrile, NaH, DMF, 90°C, 5.5 h.; (d) trans-l,4-diaminocyclohexane, NaOtBu, Pd₂(dba)₃, DavePhos, DME, 50°C, overnight; (e) DMSO, EtOH, NaOH, H₂O₂, rt, 3 h.; (f) 6-(BOC-amino)caproic acid, EDCI, DMAP, rt, 2 h; (g) TFA, CH₂C1₂, rt; (h) Affigel-10, DIEA, DMAP, DMF.

Scheme 5

Synthesis of SNX-2112. Reagents and conditions: (a) O-THP-trans-cylohexanolamine (24), NaOtBu, Pd₂(dba)₃, DavePhos, DME, 60°C, 3.5 h; (b) DMSO, EtOH, 5N NaOH, H₂O₂, rt, 4 h; (c) PPTS, EtOH, 65°C, 17 h.