Natural Product Inspired N-Terminal Hsp90 Inhibitors: From Bench to Bedside?

Abstract

The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Proteins dependent upon Hsp90 are associated with all six hallmarks of cancer. Upon Hsp90 inhibition, protein substrates are degraded via the ubiquitin-proteasome pathway. Consequentially, inhibition of Hsp90 offers a therapeutic opportunity for the treatment of cancer. Natural product inhibitors of Hsp90 have been identified in vitro, which have served as leads for the development of more efficacious inhibitors and analogs that have entered clinical trials. This review highlights the development of natural product analogs, as well as the development of clinically important inhibitors that arose from natural products.

Keywords: Hsp90; chaperone; geldanamycin; natural product-based drug design; radicicol.

Figure 1

Natural product inhibitors of Hsp90. 1-4 bind Hsp90 in the N-terminal ATP-binding pocket. 5 is an allosteric modulator of Hsp90. 6-8 disrupt the interaction of Hsp90 and co-chaperones. 9-11 bind the C-terminal ATP-binding motif.

Figure 2

The Hsp90 chaperoning cycle highlighting the effect of inhibitors on the cycle (1.6)

Figure 3

GDA analogs reported from Pfizer. IC₅₀’s determine by measuring depletion of p185 (an client protein of Hsp90) in SkBr3 cells. IC₅₀ of GDA reported at 70 nM.

Figure 4

19-20 reported by Conforma Therapeutics. IC₅₀ values were determined using a competitive binding assay measuring compounds binding to Hsp90 within the cell lysate of MCF-7 cells. The IC₅₀ of 17-AAG was found to be 20 nM. 21-22 were reported by Kosan Biosciences. IC₅₀ values were determined in SKBr3 cells and 17-AAG had a reported IC₅₀ of 33 nM.

Figure 5

GDA analogs prepared via semi-synthesis. K_d of 23-24 were measured in a competitive binding assay (17-AAG reported as 110 nM). ED₅₀ values of 25-26 were determined via Her2 degradation in SkBr3 cells (GDA reported at 5 nM). EC₅₀ of 27-30 were measured via fluorescence polarization with Hsp90α and BODIPY-GDA as a probe (17-AAG reported at 119 nM).

Figure 6

K_d of 31 was determine using Scintillation Proximity Assay for Hsp90 binding (GDA = 670 nM). K_d of 32 was determined using ITC. IC₅₀ of 33 was determined using an ATPase inhibition assay (GDA = 3.19 μM). IC₅₀ of 35 was determined in a competitive binding assay using FITC-GDA. Biological data was not reported for 36.

Figure 7

Structure of probes synthesized to assess Hsp90 biology and function.

Figure 8

Linker and 3,4,5-trimethoxycinnamyl group (41) provide decreased hepatotoxicity while maintaining efficacy similar to 17-AAG. 19-substitutions of GDA synthesized to mimic attack of biological nucleophiles, such as thiols (42-43).

Figure 9

Natural product resorcinol-based inhibitors of Hsp90 (4, 44-46). 47-49 synthetic analogs of 4 to increase in vivo stability. IC^a₅₀ represents IC₅₀ values of antiproliferative activity against the MCF-7 breast cancer (4, 44, 47-48) and the KNRK5.2 (49) cell line. IC^b₅₀ represents IC₅₀ values of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.

Figure 10

Binding modes of GDA (A) and RDC (B) to the Hsp90 N-terminal ATP-binding site. The hydrogen bonding network of the carbamate and resorcinol with the binding site has been proven to be critical for Hsp90 affinity. PDB Codes: 1A4H for GDA and 1AH6 for RDC.

Figure 11

Resorcinol containing Hsp90 inhibitors currently in clinical trials. IC₅₀ values represent antiproliferative activity against various cancer cell lines.

Figure 12

Development of the first chimeric Hsp90 inhibitor (55) and the radanamycin seco agents, radamide and radester.

Figure 13

Radamide bound to Grp94 (A) and yeast Hsp90 (B). A depicts 5′-extension pocket present in Grp94. Cis-Radamide projects towards the unique pocket of Grp94.

Figure 14

Grp94-selective inhibitors. Incorporation of a cis-amide bioisostere led to the development of BnIm. 59 represents the most active compound from the Grp94-selective radamide analogs. 60 is an analog of BnIm that has been shown to reduce mutant myocilin aggregation in vitro.

Figure 15

Structures of purine-based Hsp90 inhibitors. EC₅₀ values were determined in a competitive assay with GDA on Affi-Gel resin.

Figure 16

Second generation purine analogs with enhanced potency and solubility. IC₅₀^a values for 69-71 correspond to anti-proliferative activity in MCF-7 cells (MTS assay) and SkBr3 cells (sulforhodamine B assay) for 72. IC₅₀^b corresponds to the Her2 degradation assay 69-72 in the same cell lines used for determining anti-proliferative activity. 73-74 represent rationally designed purine analogs. 75 is a purine-based Grp94-selective inhibitor