Co-crystalization and in vitro biological characterization of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazole Hsp90 inhibitors

Abstract

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90.

Figure 1. Chemical strucures of the thiadiazole compounds and NVP-AUY922.

The K_d values for binding to Hsp90 are indicated.

Figure 2. PyMOL diagram showing binding interactions with Hsp90.

(A) Interactions of ICPD 34 and (B) ICPD 47 with the N-terminal domain of human Hsp90α. Dotted blue lines represent hydrogen bonds and the amino-acid residues involved are in green; cyan-colored spheres represent water molecules and cyan residues are amino acids solely in van der Waals contact. The structures for ICPD 34, and ICPD 47 were obtained at 2.5, and 1.4 Å resolution, respectively. ICPD 26, which lacks a 3-methoxy group relative to ICPD 34, binds in essentially the same manor as ICPD 34 and ICPD 47. For atomic coordinates and structure factors, see PDB codes 2YI0 (ICPD 26), 2YI5 (ICPD 34), and 2YI7 (ICPD 47).

Figure 3. PyMol cartoons of the structure of human Hsp90-drug complexes.

(A), PyMol cartoon showing ICPD 34 (cyan) and NVP-AUY922 (yellow and magenta) bound to the N-terminal domain of human Hsp90α. The magenta region of the NVP-AUY922-Hsp90α complex represents the unstructured region of the ICPD 34-Hsp90α complex. The mobile loop region interacting with NVP-AUY922 is shown in gold. Water molecules are shown as cyan colored spheres and hydrogen bonds as dotted blue lines. The 3-methoxy group of ICPD 34 causes the loop represented by Asn 105 to Ile 110 to adopt an alternative conformation, which moves from the gold to green conformation. (B), Two orthogonal views of the superimposition of ICPD 34 (Cyan) and NVP-AUY922 (yellow) showing that the compounds bind with overall similarity, except for the morpholino group of NVP-AUY922 and the dimethoxyphenyl group of ICPD 34. The structures for ICPD 34 and NVP-AUY922 were obtained at 2.5 and 2.0 Å resolution, respectively. For atomic coordinates and structure factors, see PDB codes 2YI5 and 2VCI, respectively.

Figure 4. The biomarker signature of Hsp90 inhibition.

Western blot showing depletion of Hsp90 client proteins and induction of Hsp72, Hsp27 and cleaved PARP, upon treatment with 1×, 5× and 10× GI₅₀ concentrations of compounds in HCT116 human colon cancer cells for 24 hr. 17-AAG was used as a positive control. Gapdh was used as loading control. Induction of cleaved PARP is also shown and is indicative of apoptosis.