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. 2024 Sep 2;15(10):3609-3615.
doi: 10.1039/d4md00501e. Online ahead of print.

Rationally modified SNX-class Hsp90 inhibitors disrupt extracellular fibronectin assembly without intracellular Hsp90 activity

Gciniwe S Mathenjwa  1   2 , Abir Chakraborty  3 , Abantika Chakraborty  3 , Ronel Muller  2 , Mathew P Akerman  2 , Moira L Bode  4 , Adrienne L Edkins  3 , Clinton G L Veale  1
Affiliations

Rationally modified SNX-class Hsp90 inhibitors disrupt extracellular fibronectin assembly without intracellular Hsp90 activity

Gciniwe S Mathenjwa et al. RSC Med Chem. .

Abstract

Despite Hsp90's well documented promise as a target for developing cancer chemotherapeutics, its inhibitors have struggled to progress through clinical trials. This is, in part, attributed to the cytoprotective compensatory heat shock response (HSR) stimulated through intracellular Hsp90 inhibition. Beyond its intracellular role, secreted extracellular Hsp90 (eHsp90) interacts with numerous pro-oncogenic extracellular clients. This includes fibronectin, which in the tumour microenvironment enhances cell invasiveness and metastasis. Through the rational modification of known Hsp90 inhibitors (SNX2112 and SNX25a) we developed four Hsp90 inhibitory compounds, whose alterations restricted their interaction with intracellular Hsp90 and did not stimulate the HSR. Two of the modified cohort (compounds 10 and 11) were able to disrupt the assembly of the extracellular fibronectin network at non-cytotoxic concentrations, and thus represent promising new tool compounds for studying the druggability of eHsp90 as a target for inhibition of tumour invasiveness and metastasis.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Examples of cell impermeable Hsp90 inhibitors (2 and 4), and their parent compounds (1 and 3).
Fig. 2
Fig. 2. Compounds 8–11 described in this study, derived from the SNX class of HSP90 inhibitory compounds 5–7.
Fig. 3
Fig. 3. The lowest energy docked binding mode of compound 11 (pink) overlaid with x-ray co-crystal structure of SNX2112 (5, blue) in complex with the N-terminal domain of Hsp90 (PDB 6LTK). The predicted binding pose suggests that compounds 8–11 retained key binding site interactions including electrostatic interactions with Tyr139, Asp93 (white) and binding site waters (red), as well as hydrophobic interactions with Leu103, Phe138, Val150 and Trp162 (green). This data indicated that our proposed structural modifications would not disrupt engagement with the SNX2112 binding pocket.
Scheme 1
Scheme 1. i) (CF3CO)2O, TNF, NEt3, 55 °C, 2 h; ii) NaOH, MeOH, H2O, r.t., 3 h, 45%; iii) 2-bromo-4-fluorobenzonitrile, NaH, anhy. DMSO, 45 °C, 40 h, 57%; iv) trans-N-Boc-1,4-cyclohexanediamine, DPPF[PdCl2], DPPF, NatOBu, THF, 65 °C, 65%; v) 2-fluoro-4-hydrazinylbenzonitrile, MeOH, AcOH, r.t., 3 d, 67%; vi) trans-N-Boc-1,4-cyclohexanediamine, DIPEA, DMSO, 90 °C, 60 min, 74%; vii) EtOH, NaOH, 30% H2O2, H2O, r.t., 16 h, 60%. viii) TFA, DCM, r.t., 5 h, 41–79%. ix) 6-Phosphonohexanoic acid, DIPEA, EDCI, NHS, DMF, r.t., 42 h, 53–62%; x) 4-bromo-1-butanesulfonic acid, DIPEA, DMF, 50 °C, 18 h, 39–52%.
Fig. 4
Fig. 4. Densitometry readings following Western blot of HeLa cells treated with compounds 5, 8–11. Treatment with validated intracellular Hsp90 inhibitor SNX2112 (5) resulted in an expected reduction in levels of Hsp90 client CDK4 (A), with a concomitant increase in Hsp70 levels (B). Treatment with modified analogues 8–11 did not mirror this same effect, indicating a lack of engagement with intracellular Hsp90.
Fig. 5
Fig. 5. Fluorescence microscopy images and fluorescence intensity quantification of fibronectin matrix assembly. This data indicates that, at sub-cytotoxic concentrations, two modified SNX analogues 10 and 11 disrupted fibronectin matrix assembly in a similar fashion to wild-type functional upstream domain (WT-FUD), a known fibronectin inhibitor.
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