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. 2020 Jun 11;11(8):1535–1538. doi: 10.1021/acsmedchemlett.0c00100

From Bacteria to Cancer: A Benzothiazole-Based DNA Gyrase B Inhibitor Redesigned for Hsp90 C-Terminal Inhibition

Kyler W Pugh †, Zheng Zhang †, Jian Wang ‡, Xiuzhi Xu ‡, Vitumbiko Munthali †, Ang Zuo †, Brian S J Blagg †,*
PMCID: PMC7429967  PMID: 32832020

Abstract

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Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for the folding and maturation of client proteins that are associated with all ten hallmarks of cancer. Hsp90 N-terminal pan inhibitors have experienced unfavorable results in clinical trials due to induction of the heat shock response (HSR), among other concerns. Novobiocin, a well characterized DNA gyrase B inhibitor, was identified as the first Hsp90 C-terminal inhibitor that manifested anticancer effects without induction of the HSR. In this letter, a library of Hsp90 C-terminal inhibitors derived from a benzothiazole-based scaffold, known to inhibit DNA gyrase B, was designed, synthesized, and evaluated. Several compounds were found to manifest low micromolar activity against both MCF-7 and SKBr3 breast cancer cell lines via Hsp90 C-terminal inhibition.

Keywords: Hsp90 inhibitors, DNA Gyrase B, Hsp90 C-terminal, benzothiazole

The 90 kDa heat shock proteins (Hsp90) remain a sought after target for the development of novel therapeutics for the treatment of cancer and/or neurodegenerative diseases.1,2 Hsp90 is a molecular chaperone that plays a pivotal role in cellular proteostasis and is responsible for the folding and/or activation of more than 300 Hsp90-depedent client substrates. Moreover, Hsp90-dependent client proteins such as Her2, Akt, Raf1, CDK4/6, Src, telomerase, and survivin are directly associated with the ten hallmarks of cancer, which further supports the development of Hsp90 inhibitors for the treatment of cancer. Hsp90 contains an N-terminal ATP-binding site, a middle domain, and a C-terminus.3,4

Historically, the focus of Hsp90 research centered on the design of inhibitors that target the N-terminal ATP-binding-site for the treatment of cancer, and this approach led to 17 therapeutic agents that underwent clinical investigation. Unfortunately, the Hsp90 N-terminal inhibitors experienced major challenges in the clinic, as induction of the pro-survival heat shock response (HSR) led to increased levels of Hsp90 and, consequently, cytostatic activity.5 During the HSR, heat shock proteins are overexpressed to maintain cell viability under conditions in which proteins are often denatured.5,6 Therefore, the discovery of novel Hsp90 inhibitors that manifest anticancer activity without induction of the pro-survival HSR is highly desired.6,16

DNA gyrase is a Type II topoisomerase that reduces the topological strain caused by the breaking and rejoining of double stranded DNA during replication. DNA gyrase is composed of two subunits (GyrA and B), whereas GyrB is the only subunit that contains an ATP-binding domain.7,8 Hsp90 and DNA gyrase B share high homology between their ATP-binding sites.11 In fact, both proteins have a unique Bergerat fold in this region that allows for the design of selective inhibitors.14,15 Novobiocin is a clinically approved DNA Gyrase B inhibitor used to treat bacterial infection by binding to the Bergerat fold present in DNA Gyrase (Figure 1). Due to similarities between DNA gyrase and Hsp90, Neckers and co-workers hypothesized that novobiocin may also inhibit Hsp90s Bergerat fold.9,10 Upon scientific investigation, novobiocin was found to inhibit Hsp90 at a previously unrecognized C-terminal binding region and to exhibit allosteric control over the N-terminal ATP-binding site. Moreover, novobiocin was identified as the first Hsp90 C-terminal inhibitor and, remarkably, was capable of Hsp90 inhibition without induction of the HSR.9,10 Unfortunately, novobiocin manifests an EC50 value of ∼700 μM against Hsp90, and no cocrystal structure of Hsp90 bound to a C-terminal inhibitor exists, which hinders the development of improved analogs.12 Consequently, it was proposed that other DNA Gyrase B inhibitors (e.g., compound 2) may also inhibit the Hsp90 C-terminus.

Figure 1.

Figure 1

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Structures of DNA Gyrase B and Hsp90 inhibitors. (1) Novobiocin, a DNA Gyrase B and Hsp90 C-terminal inhibitor. (2) Benzothiazole-based DNA Gyrase B inhibitor.

Results and Discussion

Two novel Hsp90 C-terminal scaffolds, A and B, were selected (Figure 2) from a series of benzothiazole-based DNA Gyrase B inhibitors discovered by Kikelj and co-workers (Figure 1, compound 2).7,13 Recently, DNA Gyrase B inhibitors were identified that manifest anti-influenza activity through Hsp90 inhibition, although no data was provided to clarify where the compounds bound Hsp90.17 In this study, compounds 3 and 4 were shown to manifest antiproliferative activity against breast cancer cell lines, and compound 4 was demonstrated to inhibit Hsp90 via the C-terminus (see Figure 1S). Therefore, structural diversification of these scaffolds was sought and included (1) modifications to the ethyl oxalyl substituent and/or (2) modifications/substitutions to the pyrrole.

Figure 2.

Figure 2

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Benzothaizole-based DNA Gyrase B inhibitors reveal two novel scaffolds for Hsp90 C-terminal inhibition.

Replacements for the ethyl oxalyl substituent included an acetamide, oxopropanamide, and 2-oxo-phenylacetamide. The acetamide group was selected to probe the substituents that are necessary for activity, whereas investigation of the electrophilic properties of the ethyl oxalyl moiety was undertaken via replacement with an oxopropanamide or 2-oxo-2-phenylacetamide. A variety of aromatic moieties were also proposed as surrogates for the pyrrole ring and included indole derivatives, mono- and dibromo pyrroles, dimethyl pyrrole, phenyl[1,3]dioxole, phenyl, and pyrimidine moieties.

In this study, two general synthetic schemes were utilized to prepare these scaffolds (Schemes 1 and 2). Commercially available 6-nitrobenzo[d]thiazol-2-amine (5) was coupled with ethyl oxalyl chloride (6) to form intermediate 7. The nitro group was reduced to the aniline (intermediate 8) via a Pd-catalyzed hydrogenolysis under a hydrogen atmosphere. Subsequent amide coupling provided compound 3, as well as related analogs (Scheme 1). Synthesis of analog 4 and scaffold B derivatives followed a similar synthetic scheme (Scheme 2). Commercially available 6-nitrobenzo[d]thiazol-2-amine (5) was coupled with acid chloride 9 to form intermediate 10. The nitro group was reduced to the aniline (intermediate 11) by Pd-catalyzed hydrogenolysis under a hydrogen atmosphere. Subsequent amide coupling provided compound 4, as well as related analogs.

Scheme 1. Synthesis of Scaffold A.

Scheme 1

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Reagents and conditions: (a) Pyridine, rt, 12 h; (b) H2, 10% Pd/C, MeOH and THF, rt, 12 h; (c) DCM, Et3N, rt, 12 h.

Scheme 2. Synthesis of Scaffold B.

Scheme 2

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Reagents and conditions: (a) DCM, Et3N, rt, 12 h; (b) H2, 10% Pd/C, MeoH and THF, rt, 12 h; (c) pyridine, rt, 12 h.

Several of the compounds displayed low μM EC50 values against breast cancer cell lines. For example, compounds 3 and 4 manifested EC50 values of 3.59 μM and 2.41 μM against the SKBr3 breast cancer cell line, respectfully (Tables 1 and 2). Compound 3c was synthesized to contain a 4-bromo-pyrrole substituent, but activity for this analog was decreased ∼8-fold as compared to lead compound 3. Additional substitutions to the R1 side chain of scaffold A were pursued, but those compounds produced similar results. Compounds 3b and 3d were also found to decrease antiproliferative activity; however, analogs derived from scaffold B continued to show reasonable activity against both SKBr3 and MCF-7 breast cancer cells. Based on the data collected, we shifted our focus to the preparation of scaffold B analogs due to their greater inhibitory activities. (Table 2).

Table 1. Scaffold A Analogs.

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Table 2. Scaffold B Analogsa.

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a

Compounds with calculated std dev tested N = 3, w/o N = 1.

Replacement of the ethyl oxalyl group with the 2-oxo-2-phenylacetamide or an acetamide moiety on scaffold B resulted in compounds 4a–c, which were devoid of activity (Table 2). Antiproliferative data produced by 4d demonstrated that substitutions on the indole ring led to increased inhibitory activity when a methyl group was placed at the 5-position, as compared with the unsubstituted analog, 4i. SAR for the 5-position of the indole moiety was further explored and additional analogs prepared. This approach led to the discovery of lead compound 4e, which exhibited an EC50 value of 0.86 μM and 7.03 μM against SKBr3 and MCF-7 breast cancer cell lines, respectively (Table 2). Using the EC50 data manifested by 4e and 4d, we proposed that an increase in electron density on the indole could contribute to an increase in antiproliferative activity. Although it is also possible that the addition of a hydrogen bond acceptor may increase inhibitory activity. Consequently, compound 4f was synthesized to include an ethyl moiety at the 5-position. The activity exhibited by 4f was diminished ∼20-fold and supported our previous hypothesis that electron donating groups increase potency for these analogs. Therefore, a compound was synthesized to contain a methoxy group at the 6-position to identify the optimal position for inclusion of an electron donating group (4g). The EC50 manifested by this compound increased to ∼7 μM, which when compared to 4e demonstrated that electron donating or hydrogen bonding groups at the 5-position are more effective. The antiproliferative activities manifested by these compounds were shown to result from Hsp90 inhibition, as demonstrated via Western blot analysis of SKBr3 cell lysates following the administration of compound 4e (Figure 3), as Hsp90-dependent substrates were degraded without induction of Hsp70 levels.

Figure 3.

Figure 3

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Western blot analyses from SKBr3 lysates after administration of compound 4e after 24 h. [GDA] = 500 nM. Image quantification provided in the Supporting Information.

Conclusion

Two novel Hsp90 C-terminal inhibitory scaffolds were identified from a series of DNA Gyrase B inhibitors. Compound 4 was shown to induce Hsp90 client protein degradation without induction of the HSR, a hallmark of Hsp90 C-terminal inhibition. Therefore, a library of analogs was prepared and evaluated against MCF-7 and SKBr3 breast cancer cell lines. Several analogs were shown to manifest potent antiproliferative activity against these breast cancer cells. However, scaffold B compounds demonstrated greater inhibitory activity than scaffold A, and therefore, SAR studies were pursued on scaffold B. It was determined that compound 4e manifested the greatest potency and remains a lead compound for future studies aimed at elucidating structure–activity relationships for benzothiazole-based Hsp90 C-terminal inhibitors. The results from such studies will be reported in due course.

Acknowledgments

Financial support of the project was provided by the NIH (CA120458).

Glossary

Abbreviations

Hsp90

heat shock protein 90

HSR

heat shock response

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00100.

  • Synthetic experimental details, characterization of compounds, and biological data (PDF)

Author Contributions

§ K.W.P. and Z.Z. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ml0c00100_si_001.pdf (446.2KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml0c00100_si_001.pdf (446.2KB, pdf)

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