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. 2024 Apr 8;15(5):619–625. doi: 10.1021/acsmedchemlett.4c00022

Discovery of Disubstituted Carboranes as Inhibitors of Heat Shock Protein 90–Heat Shock Factor 1 Interaction

Yujie Shao †, Kazuki Miura †,‡, Yasunobu Asawa †, Taiki Morita †,‡, Guangzhe Li §, Hiroyuki Nakamura †,‡,*
PMCID: PMC11089554  PMID: 38746882

Abstract

graphic file with name ml4c00022_0009.jpg

Efficient synthesis of disubstituted para- and ortho-carboranes (2 and 3, respectively) was achieved. Among the compounds synthesized, 3e showed potent suppression of hypoxia-inducible factor 1 (HIF-1) transcriptional activity under hypoxia by a cell-based reporter gene assay. Detailed mechanism-of-action studies revealed that 3e reduced the stability of heat shock protein (HSP) 90 client proteins such as CDK4, AKT, and cyclin D1 by inhibiting HSP90 chaperone activity but did not induce a heat shock response (HSR), which may cause drug resistance. Furthermore, 3e inhibited the interaction between HSP90 and heat shock factor 1 (HSF1), resulting in reducing HSF1 protein stability and thereby suppressing the transcription of heat shock proteins.

Keywords: Carborane, Hypoxia-inducible factor 1, Heat shock protein 90 inhibitor, Heat shock factor 1

Heat shock protein (HSP) 90 is an evolutionarily conserved oncogenic target protein that regulates multiple client proteins involved in constitutive cell signaling pathways and adaptive response to stresses such as heat and hypoxia.1,2 Inhibition of HSP90 leads to the degradation of these client proteins, resulting in the disruption of multiple signaling pathways essential for tumor cell proliferation, survival, and angiogenesis; thus HSP90 has been considered an attractive target for cancer therapy.2,3 HSP90 inhibitors developed to date have been classified according to their binding domains (Figure 1).4,5 The initial efforts to explore HSP90 inhibitors focused on competitors that bind to the N-terminal ATP pocket, such as geldanamycin (GA)6 and radicicol.7 However, these competitors were unsatisfactory in terms of efficacy in cancer therapy because they up-regulate the expression of other HSPs through induction of heat shock response (HSR), which may cause drug resistance.8 In 2022, pimitespib, an oral HSP90 inhibitor, was approved in Japan as the world’s first HSP90-targeted anti-cancer drug for the treatment of gastrointestinal stromal tumors.9 The co-crystal structure of HSP90 with a pimitespib analogue disclosed a unique binding mode at the N-terminal ATP pocket distinct from typical known HSP90 N-terminal inhibitors.10 Nevertheless, pimitespib cannot avoid HSR induction, causing up-regulation of HSP70.11 Therefore, a novel type of HSP90 inhibitor that does not induce HSR is required for HSP90-targeted cancer therapy. The second target of HSP90 inhibitors is protein–protein interactions (PPIs) in HSP90-induced complexes. For example, celastrol has been reported to disrupt the PPI between HSP90 and CDC37 and induce the degradation of HSP90 client proteins such as CDK4 and AKT while increasing the expression of HSP70.12 However, PPI inhibition of HSP90 complexes causes HSR-induced upregulation of HSPs, similar to that of the inhibitors targeting the N-terminal ATP pocket. The third target of HSP90 inhibitors is the HSP90 C-terminal domain, which contains a dimerization motif and a binding site for co-chaperones. Dimerization via this motif plays an important role for HSP90 to function as a chaperone molecule.1,2 Recently, HSP90 inhibitors targeting the C-terminal domain, such as SM122 and KU135, have been developed and shown to avoid the HSR induction.13,14 Therefore, HSP90 inhibitors targeting the C-terminal domain have potential as HSP90-targeted cancer therapeutics that can avoid HSR activation and drug resistance.

Figure 1.

Figure 1

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Structure of typical HSP90 inhibitors.

Carborane is an icosahedral structure consisting of 2 carbon atoms, 10 boron atoms, and 12 hydrogen atoms, providing three different isomers, ortho, meta, and para, depending on the relative position of two carbons.15 Its unique physical and chemical properties make it a promising three-dimensional scaffold for the development of small molecular drugs.16 Various drug candidates have been reported, including estrogen receptor agonists,17 HSP60 inhibitors,18−20 COX-2 inhibitors,21 and vitamin D receptor ligands.22 We previously constructed a compound library consisting of tri- or disubstituted carboranes with benzyl and i-butyl groups as Phe and Leu mimics respectively, based on chemical space exploration.23 In this library, several di- and trisubstituted para-carboranes, such as 1 and 2a (Figure 2), showed potent inhibitory activity against the intracellular transcription of hypoxia-inducible factor 1 (HIF-1). However, the action mechanism of these carboranes to inhibit the HIF-1 transcription remains to be elucidated.

Figure 2.

Figure 2

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Design of carboranes with HIF-1 inhibitory activity.

In this study, we focused on disubstituted para-carborane 2a. Since ortho-carborane is more readily available at a lower cost than para-carborane, we established an efficient synthesis of the disubstituted ortho-carborane 3a as a mimic of the para-carborane 2a (Figure 2). Interestingly, some of the disubstituted ortho-carboranes 3a showed higher inhibitory activity against HIF-1 transcriptional activity than the trisubstituted carborane 1. Further detailed studies revealed that the disubstituted carboranes selectively bind to HSP90 and reduce the stability of HSP90 client proteins, such as heat shock factor 1 (HSF1). These observations have not been demonstrated with the previously investigated HSP90 inhibitors targeting the C-terminal domain.

The synthesis of disubstituted para-carboranes 2a and 2b is shown in Scheme 1. Starting with para-carborane, electrophilic iodination at the B2-position followed by Sonogashira coupling with trimethylsilylacetylene gave compound 5 quantitatively.24,25 Lithiation of 5 with n-BuLi followed by addition to paraformaldehyde gave the corresponding hydroxyl methylated disubstituted para-carborane 6 in 39% yield, together with the other isomer 6′ (38%). After deprotection of the trimethylsilyl (TMS) group of 6 with K2CO3, the resulting hydroxy group was protected with a tert-butyldimethylsilyl (TBS) group to afford compound 7 in 91% yield in 2 steps. Lithiation of 7 with n-BuLi followed by addition to CO2 introduced a carboxylic acid moiety at the terminal alkyne position of 7 quantitatively. Condensation reaction of the resulting carboxylic acid 8 with benzyl amine and removal of the TBS group followed by esterification with iBuCOCl gave compound 9, which underwent the click cycloaddition reaction with benzyl azide to give 2a in 65% yield in 4 steps. For further derivatization, 2a was hydrolyzed under basic conditions, and the resulting alcohol 10 was oxidized using Dess–Martin reagent. The resulting aldehyde 11 was subjected to Pinnick–Kraus oxidation to give the corresponding carboxylic acid 12, which underwent a condensation reaction with isoamylamine to give 2b.

Scheme 1. Synthesis of Disubstituted para-Carboranes.

Scheme 1

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Reagents and conditions: (a) I2, HNO3, H2SO4, AcOH, 80 °C. (b) TMS-acetylene, PdCl2(PPh3)2, CuI, toluene/piperidine, 130 °C, quant. for 2 steps. (c) (CHO)n, n-BuLi, THF, −78 °C to r.t., 39%. (d) (i) K2CO3, MeOH, r.t., (ii) TBSOTf, lutidine, DCM, r.t.; 91% for 2 steps. (e) CO2, n-BuLi, THF, −78 °C to r.t., quant. (f) (i) EDCI, HOBt, BnNH2, DIEA, THF, r.t.; (ii) HCl, dioxane/DCM; (iii) iBuCOCl, EDCI, DMAP, DCM, r.t., 65% for 3 steps. (g) BnN3, toluene, 90 °C, overnight, quant. (h) NaOH, MeOH, r.t., 24 h, 40%. (i) Dess–Martin periodinane, DCM, 0 °C, 90%. (j) 2-methyl-2-butene, acetone, NaH2PO4, NaClO2, 0 °C to r.t., 80%. (k) EDCI, HOBt, isoamylamine, r.t., 86%.

Next, we aimed to synthesize disubstituted ortho-carboranes. Initially, we examined the copper-mediated coupling of C-lithiated ortho-carborane with bromoethynyl(trimethyl)silane based on the literature procedure.26 However, the corresponding C-alkynylated product was not obtained. Consequently, we conducted the synthesis of ortho-carboranes 3 through the Kumada–Tamao–Corriu cross coupling of boron-iodinated ortho-carborane with alkynylmagnesium bromide, as shown in Scheme 2. Selective iodination at the B3-position of ortho-carborane was performed via reconstruction of the ortho-carborane cage 13 with BI3 according to a literature procedure,27 and the resulting iodo-ortho-carborane 14 was subjected to the Kumada–Tamao–Corriu cross coupling to give compound 15.25,28 Hydroxymethylation of 15 was performed with paraformaldehyde in the presence of tetrabutylammonium fluoride (TBAF) to obtain the alcohol 16,29 and the resulting hydroxy group of 16 was protected with a TBS group. Since the C–H acidities of a terminal alkyne (pKa = 25) and ortho-carborane (pKa = 23) are different, the amide groups were successfully introduced to the terminal alkyne of 17 after protecting the C–H of the ortho-carborane with a TMS group.30 Without any purification, both the TMS and TBS groups were removed using TBAF to afford compounds 18 and 19 in moderate yields in 3 steps. Similarly, esterification with the corresponding acyl chlorides followed by the click cycloaddition with the corresponding azides gave the desired disubstituted ortho-carboranes 3a and 3c–h (27–97% yields).31 Furthermore, hydrolysis of 3a followed by esterification with phenylacetyl chloride afforded 3b in 46% yield in 2 steps.

Scheme 2. Synthesis of Disubstituted ortho-Carboranes.

Scheme 2

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Reagents and conditions: (a) KOH, EtOH, reflux, then HCl, Me3N·HCl, 4 h, 89%. (b) n-BuLi, Et2O, 0 °C to reflux, then BI3, toluene, r.t., overnight, 72%. (c) TMSC2MgBr, THF, Pd(PPh3)2Cl2, 50 °C, 5 h, quant. (d) TBAF, THF, (CHO)n, r.t., 10 min, 85%. (e) 2,6-Lutidine, TBSOTf, DCM, 0 °C, 2 h, 95%. (f) (i) LHMDS, THF, TMSCl, −78 °C, 1 h; (ii) LHMDS, benzyl isocyanate or isopropyl isocyanate, THF, −78 °C, 1 h; (iii) TBAF, THF, 0 °C to r.t., 2 h. (g) Isovaleryl chloride or phenylacetyl chloride, Et3N, DCM, 0 °C, 1 h. (h) iBuN3 or BnN3, toluene, 90 °C, overnight. (i) MeOH, NaOH, r.t., 1 h, 63%. (j) Phenylacetyl chloride, TEA, DCM, 0 °C, 3 h, 73%.

To elucidate the effect of carboranes on HIF-1 transcriptional activity and cancer cell proliferation, we next performed a cell-based reporter gene assay using hypoxia response element (HRE)-dependent luciferase-transfected HeLa cells23 and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, respectively.

The results are shown in Table 1. Disubstituted para-carborane 2a and disubstituted ortho-carboranes 3a, 3b, 3d, and 3e exhibited potent inhibitory activity against HIF-1 transcription, whereas 3c and 3e–h showed no inhibitory activity against HIF-1 transcription at 30 μM. Because significant cytotoxicity was observed with disubstituted para-carborane 2b, its effect on HIF-1 transcription was not investigated. We also examined the cytotoxicity of the compounds after 12 h of incubation and confirmed that the inhibitory activity of the compounds on HIF-1 transcription was independent of cytotoxicity (Table S1). Moreover, disubstituted ortho-carboranes 3a–h showed moderate inhibitory activity on cancer cell proliferation after 72 h of incubation. Among the compounds synthesized, 3e showed the most potent inhibition of HIF-1 transcription (Table 1, also see Figure S1). Subsequently, the effects of 3e on the expression of HIF-1α and its downstream proteins were examined. HeLa cells were treated with increasing concentrations of compound 3e for 8 h under hypoxia. The expression levels of HIF-1α and its downstream proteins, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF),32 were measured by Western blot analysis. As shown in Figure 3A, 3e suppressed HIF-1α protein accumulation in a concentration-dependent manner, consistent with the results for HIF-1 transcriptional inhibitory activity shown in Table 1. Furthermore, we determined the gene expression levels of HIF-1α and its downstream signal to confirm whether 3e affected the expression of these genes. As shown in Figure 3B, although 3e did not affect HIF-1α mRNA expression, mRNA expression of its downstream genes, CA9, GLUT1, and EPO, was suppressed by this compound, indicating that 3e can efficiently inhibit the HIF-1 signaling pathway.

Table 1. Effects of Disubstituted para- and ortho-Carboranes (2a, 2b, 3a–3h) on Inhibitory Activities of HIF-1 Transcription (HRE-Luc) and Cell Proliferation in HeLa Cells.

  IC50 [μM]a
Compd HRE-Lucb Cell proliferationc
2a 5.17 ± 0.55 7.08 ± 0.42
2b N.D.d 1.88 ± 0.48
3a 5.19 ± 1.52 15.43 ± 0.72
3b 3.58 ± 1.75 13.03 ± 0.13
3c >30 13.09 ± 0.67
3d 3.16 ± 0.37 14.89 ± 0.44
3e 2.16 ± 0.13 20.26 ± 0.96
3f >30 30.82 ± 0.17
3g >30 25.22 ± 2.59
3h >30 45.29 ± 4.05
1 5.35 ± 1.80 >100
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a

Indicated half-maximal inhibitory concentration (IC50) is mean ± SD of a single experiment conducted in triplicate.

b

HRE-Luc expression was measured by luciferase reporter gene assay using HRE-dependent luciferase-transfected HeLa cells incubated with compounds for 12 h under hypoxic conditions (1% O2 and 5% CO2).

c

Cell proliferation was measured by MTT assay against HeLa cells incubated with compounds for 72 h.

d

Not determined due to its high cytotoxicity.

Figure 3.

Figure 3

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Degradation of the HIF-1α protein induced by 3e. (A) Effects of 3e on HIF-1α and its downstream proteins VEGF and CA9. HeLa cells were treated with various concentrations of 3e for 8 h under hypoxia. Cell lysates were separated by SDS-PAGE, and immunoblotting was performed using HIF-1α, VEGF, CA9, and α-tubulin antibodies. Tubulin was used as an internal control. (B) Effects of 3e on mRNA expression levels of HIF-1α and its downstream genes, CA9, GLUT1, and EPO. HeLa cells were treated with various concentrations of 3e for 8 h under hypoxia. Total RNA was isolated from each cell, and mRNA was converted to cDNA. Gene expression levels were detected using real-time PCR. Each condition of hypoxia was defined as 1.00. Data are presented as means ± the standard deviation (SD) of three separate experiments. Error bars, SD. Significance was determined as *P < 0.05, **P < 0.01.

HSP90 is a chaperone protein that stabilizes a variety of client proteins, and HIF-1α is stabilized and activated by interaction with HSP90 under hypoxia.33 HSP90 forms a chaperone complex with HSP70 and HSF1 to stabilize client proteins such as CKD4, AKT, and cyclin D1.34 Consequently, the inhibition of HSP90 reduces the stability of these proteins. To evaluate the effects of 3e on HSP90, we treated HeLa cells with 3e for 12 h and evaluated the expression levels of HSP90 and its client proteins through Western blot analysis. GA, a well-known HSP90 inhibitor that reduces client protein stability and activates the HSF1 pathway by inhibiting the ATPase activity of HSP90, was used as a positive control. As expected, treatment with GA reduced the protein level of CDK4, an HSP90 client protein, and increased the protein expression of HSP70, an HSF1 downstream target protein (Figure 4A). In contrast, treatment with 3e reduced the protein level of HSP90 client protein similarly to GA and also reduced the protein levels of HSP70 and HSF1 (Figure 4B). These results suggested that 3e inhibited HSP90 through a mechanism of action different from that of GA. Furthermore, to understand the effect of 3e on the HSF1 signaling pathway, we evaluated the expression levels of HSF1-induced genes by real-time PCR. HSF1 functions as a unique transcriptional factor, and activation of the HSF1 pathway triggers upregulation of gene expression of HSPs, such as HSP70 and HSP47.35 Consistent with the literature,13,14 treatment with GA promoted the gene expression of HSF1 target genes, HSP70 and HSP47. In contrast, treatment with 3e suppressed HSP70 and HSP47 gene expression without inducing HSR through the HSF1 pathway, as shown in Figure 4C. Moreover, 3e inhibited transcriptional enhancement of HSF1 downstream genes induced by heat shock (Figure S2). These results indicate that the mechanisms of HSP90 inhibition by GA and 3e are different and that 3e may be a novel type of inhibitor that does not induce HSF1-promoted HSR.

Figure 4.

Figure 4

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Effects of GA and 3e on the expression levels of HSP90 client proteins (A and B) and the HSF1 signaling pathway (C). (A and B) HeLa cells were treated with different concentrations of GA or 3e for 12 h. Cell lysates were separated by SDS-PAGE, and immunoblotting was performed using HSP90, HSP70, CDK4, HSF1, AKT, p-AKT (T308), cyclin D1, β-actin, and α-tubulin antibodies. β-Actin and α-tubulin were used as internal controls. (C) HeLa cells were treated with 3e (50 μM) or GA (40 μM) for 12 h. Total RNA was isolated from each cell, and mRNA was converted to cDNA. Gene expression levels were detected using real-time PCR. Each condition of DMSO group was defined as 1.00. Data are presented as means ± SD of three separate experiments. Error bars, SD. Significance was determined as ***P < 0.001.

To further investigate whether 3e regulates HSF1 stability, we performed a cycloheximide (CHX) chase assay, which measures the half-life of proteins in cells.36 HeLa cells were treated with CHX or CHX and 3e, and the time-dependent HSF1 protein levels were evaluated by Western blot analysis. The results are shown in Figure 5. The half-life of HSF1 protein in HeLa cells treated with CHX was approximately 12 h, whereas that in cells treated with CHX and 3e was approximately 6 h. Moreover, to determine the degradation pathway of HSF1 by 3e treatment, we examined whether treatment with the proteasome inhibitor MG132 suppressed HSF1 degradation. The results showed that HSF1 degradation by 3e was inhibited in a MG132 concentration-dependent manner (Figure S3A). These results suggested that 3e shortened the half-life of the HSF1 protein and promoted its degradation via the ubiquitin–proteasome degradation pathway in cells. Furthermore, we evaluated HSF1 phosphorylation and subcellular localization to determine the effect of 3e on HSF1 function. Phosphorylation of Ser303/307 inactivates HSF1 by inhibiting its nuclear translocation and trimer formation for HSR induction.37 We first evaluated the phosphorylation of Ser303/307 in HSF1. This result showed that 3e treatment increased the protein level of phosphorylated HSF1 in a concentration-dependent manner (Figure S3B). Next, to evaluate the effects of 3e on the subcellular localization of HSF1, we performed immunofluorescence experiments. Most of the HSF1-derived fluorescence signals were observed in the cytoplasm under 3e-treated conditions, whereas its fluorescence signals were observed in the nucleus under GA-treated conditions (Figure S3C). These results indicated that 3e is an HSP90 inhibitor that does not induce HSR by promoting phosphorylation for HSF1 inactivation and inhibiting HSF1 nuclear translocation.

Figure 5.

Figure 5

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Time-dependent HSF1 degradation by 3e. HeLa cells were treated with 100 μg/mL cycloheximide (CHX) in the presence or absence of 3e (30 μM) for the indicated times. Cell lysates were separated by SDS-PAGE, and immunoblotting was performed using anti-HSF1 and anti-GAPDH antibodies. GAPDH was used as an internal control. Each control was defined as 1.00.

Since 3e showed the potential to inhibit HSP90 in cells, we next examined whether 3e inhibited HSP90 in vitro. Human recombinant HSP90 was prepared according to the previously reported protocol,38 and the HSP90 inhibitory activity of 3e was measured by a protein refolding assay.36 Heat-denatured firefly luciferase was incubated with recombinant HSP90 in the presence of GA and 3e, and the activity of the refolded luciferase was measured. As shown in Figure 6A, treatment with GA or 3e inhibited the refolding of heat-denatured luciferase by HSP90. We also confirmed that these compounds did not inhibit luciferase activity (Figure S4), indicating that 3e inhibited HSP90. In addition, 3e was confirmed to bind to HSP90 using a fluorescence quenching assay,39 which utilizes the phenomenon that fluorescence derived from tryptophan residues decreases when the compound interacts with proteins (Figure S5). HSF1 acts as a client protein of HSP90 to regulate HSR that controls the transcription of downstream genes.40 Therefore, we conducted immunoprecipitation experiments to elucidate the regulatory mechanism of 3e on the HSP90-HSF1 complex. As shown in Figure 6B, HSF1 was associated with HSP90 in the absence of 3e, while the association of HSF1 was drastically reduced in the presence of 3e. These results suggested that 3e inhibited the interaction between HSP90 and HSF1, resulting in degradation of HSF1. On the other hand, 3e did not induce destabilization of FKBP52 and HOP, the C-terminal binding client proteins of HSP90 other than HSF1 (Figure S6). These results suggested that compound 3e specifically inhibits the interaction between HSP90 and HSF1.

Figure 6.

Figure 6

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Effects of 3e on renaturation of heat-denatured luciferase and HSP90-HSF1 interaction. (A) Heat-denatured firefly luciferase was incubated with recombinant HSP90 protein in the presence of DMSO, GA (100 μM), or 3e (100 μM) for 30 min. The luciferase activity was measured using a luciferase assay system (Promega), and luminescence from luciferase was measured using a microplate reader. Error bars, SD; **P < 0.01. (B) HeLa cells lysate was used for immunoprecipitation. After the cell lysate was incubated with or without 3e (100 μM), the HSP90-HSF1 protein complex was pulled down using anti-HSP90 antibody. Immunoblotting of the resulting samples was performed using anti-HSP90 and anti-HSF1 antibodies. HSP90 was used as an internal control.

In conclusion, we developed a novel HSP90 inhibitor based on the disubstituted ortho-carborane scaffold that demonstrated significant inhibitory activity against the HIF-1 signaling pathway under hypoxia. HSP90 was identified as its potential target, and the suppression of HSP90 downregulated several proteins associated with tumor progression. Compound 3e bound to HSP90 and disrupted the interaction between HSP90 and HSF1, resulting in reduced HSF1 protein stability and suppression of HSPs transcription and HSR. Validation with compound 3e represents a major advance in our understanding of HSP90 regulation and opens new avenues for therapeutic intervention targeting the complex network of HSP-related pathways. More optimization and investigations are ongoing for figuring out the binding domain of disubstituted carboranes and their effects on GA-resistant cancer cell lines, aiming for much more efficient novel HSP90 inhibitors exploration.

Acknowledgments

We thank Dr. Yoshihisa Sei (Open Facility Center, Tokyo Institute of Technology) for technical assistance with X-ray crystallographic analysis and the Open Research Facilities for Life Science and Technology, Tokyo Institute of Technology, for the real-time PCR analysis. This work was partially supported by JSPS KAKENHI Grant Number JP22K19104 and Chugai Foundation for Innovative Drug Discovery Science: C-FINDS.

Glossary

Abbreviations

GA geldanamycin GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HIF-1

hypoxia-inducible factor 1

HRE

hypoxia response element

HSP

heat shock protein

HSF1

heat shock factor 1

VHL

von Hippel–Lindau

PCR

polymerase chain reaction

VEGF

vascular endothelial growth factor

CA9

carbonic anhydrase IX

GLUT1

glucose transporter 1

EPO

erythropoietin

CDK4

cyclin-dependent kinase 4

AKT

protein kinase B

p-AKT

phospho-AKT

CHX

cycloheximide

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00022.

  • Synthetic procedures and details of compound characterization (1H NMR, 13C NMR, and 11B NMR), X-ray crystallographic analysis of compound 2b, and biological procedures (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml4c00022_si_001.pdf (6.8MB, pdf)

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