Research paper
Development of GLUT4-selective antagonists for multiple myeloma therapy

https://doi.org/10.1016/j.ejmech.2017.08.029Get rights and content

Highlights

  • A series of novel GLUT4 antagonists were designed and synthesized.

  • Lead compounds have high selectivity for GLUT4 versus other GLUT isoforms.

  • Compound 20 inhibits GLUT4-mediated glucose uptake and MM cell proliferation.

  • Treatment of patient samples with compound 20 causes sensitization to MM drugs.

Abstract

Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.

Introduction

Tumor cells, including those of the largely fatal plasma cell malignancy multiple myeloma (MM), exhibit elevated glucose uptake [1], [2], [3], a cancer hallmark that forms the basis for clinical monitoring of myeloma using positron emission tomography (FDG-PET) [3], [4], [5], [6]. Although the fundamental reliance of tumor cells on increased glucose catabolism for survival, proliferation and chemoresistance is well established, therapeutic targeting of this key metabolic phenotype in a tumor-specific manner has not yet been achieved [7], [8]. In addition, despite the broad relevance of elevated glucose uptake in cancer cells, there is limited understanding of the transporters responsible for enabling glucose uptake in distinct tumor contexts. Previously, we determined that constitutive plasma membrane localization of the glucose transporter isoform 4 (GLUT4), plays a critical role in sustaining glucose metabolism and the proliferation and survival of myeloma cells [9], [10].

The insulin-responsive facilitative glucose transporter GLUT4 exhibits limited tissue specific distribution and is most abundantly expressed in muscle and adipose tissue [11], [12]. GLUT4 is normally retained within the cell, trafficking to the plasma membrane (PM) upon insulin, exercise or specific stimulation that engage the PI3K/AKT [13] or AMPK [14] pathways to recruit GLUT4 to the plasma membrane [15]. In contrast, the more ubiquitously expressed isoform GLUT1 is constitutively localized to the PM facilitating basal glucose transport. GLUT1 activity is critical for the normal function of several cell types including erythrocytes, neurons, and endothelial cells thereby accounting for transport of glucose across the blood-brain barrier [16], [17]. We reported that MM cells surprisingly exhibit increased constitutive expression of GLUT4 on the PM, co-opting use of this transporter (among the 14 GLUTs) and not GLUT1 for survival and proliferation [9], [10], [18]. In addition, it was also shown that GLUT4 inhibition abrogates cell proliferation and chemoresistance in vitro in MM, chronic lymphocytic leukemia (CLL), solid tumor lines and in vivo in a xenograft model of MM [9], [10], [18], [19]. Roles for GLUT4 have also been suggested in human gastrointestinal tumors that exhibit enhanced PM localization of GLUT4 and weak expression of GLUT1 [20] and in breast cancers [21]. In sum, these observations suggest GLUT4 serves a unique role in both solid and liquid cancers.

The effects of GLUT4 knockdown were recapitulated by treatment with the HIV protease inhibitor ritonavir, a known GLUT4 antagonist (Fig. 1) [22]. The affinity of ritonavir for GLUT4, however, is in the low micromolar range [23]. Furthermore, ritonavir also exhibits inhibitory activity against GLUT1 [23]. Efforts to develop HIV protease inhibitors devoid of GLUT4 affinity have demonstrated that the modified tripeptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe, Ki 26 μM) mimics the core structure of ritonavir and is sufficient to selectively inhibit GLUT4 over GLUT1 [24]. The HIV protease inhibitor indinavir also has significant inhibitory activity towards GLUT4 [23], [25]. Others have developed glucose transporter inhibitors effective against a range of GLUT isoforms, including GLUT1 [26]. However, most of these compounds have had relatively modest potency at inhibiting GLUT4 and importantly have lacked selectivity for this isoform.

To generate more potent, non-competitive, reversible, and isoform-selective GLUT4 inhibitors, we previously generated an in silico homology model for GLUT4 and screened a library of eighteen million compounds [27]. Despite 68% homology between GLUT1 and GLUT4, a virtual screen identified two novel compounds, compound 3 and compound 17, (and related analogs 26 and 39 were also identified) that target GLUT4 selectively [27]. Importantly, modeling suggests that these inhibitors interact with critical residues of GLUT4 (Asn176 and Ile42) known to confer selectivity of HIV protease inhibitors for inhibiting GLUT4 over GLUT1 [28]. These promising results suggest it may be possible to selectively inhibit GLUT4, and thereby produce agents that specifically target cancer cells that rely on glucose transport via GLUT4.

Despite the introduction of new therapeutics, MM remains incurable in a majority of patients due to the development of resistance linked to the inability to induce apoptosis [29], [30], [31], [32]. Targeting GLUT4 in MM leads to apoptosis in MM cells associated with suppression of the resistance promoting BCL-2 family member MCL-1 [9], [33]. MM cells resistant to the cytotoxic effects of GLUT4 inhibition were found to induce chemosensitizing alterations in BCL-2 proteins, supporting the use of GLUT4 inhibitors as both therapeutic agents and chemosensitizers. The development of potent GLUT4 inhibitors will allow us to further elucidate glucose sustained metabolic and signaling sequelae that sustain survival in MM. These observations form the basis for the rationale that optimized GLUT4 inhibitors will offer unique tools and drug discovery leads to study and target glucose metabolism sustained by GLUT4 both in vitro and in vivo. Here, we report our initial medicinal chemistry efforts at developing our lead series of GLUT4 antagonists into more potent and selective inhibitors.

Section snippets

Antagonist synthesis

In an earlier screen for specific GLUT4 inhibitors, compound 26 was identified which had an IC50 value for inhibition of glucose transport through GLUT4 at 1.7 μM and >100 μM for GLUT1 [27]. However, compound 26 also inhibits glucose transport through GLUT8 at an IC50 of 6 μM [27]. Therefore, we wanted to develop more specific GLUT4 inhibitors with lower affinity for GLUT1 and GLUT8. Based on the structure of this hit series and molecular modeling, we determined that the tertiary amide and the

Discussion

Glucose uptake in tumor cells is a key rate-limiting step in glucose metabolism [37]. GLUT1 is elevated in a number of cancers; however, being widely expressed and a major glucose transporter in neurons, erythrocytes, endothelial cells and consequently the blood brain barrier [16], [17], it undermines the utility of targeting GLUT1 for cancer therapy. GLUT4 is expressed in muscle (skeletal and heart) and adipose tissue and plays a central role in whole body glucose homeostasis by facilitating

Conclusion

We have carried out intiial hit-to-lead medicinal chemistry on our novel series of GLUT4 antagonists. These analogs explored several areas of the scaffold and initial structure-activity relationships were identified. Our lead compound possesses excellent selectivity for GLUT4 over other GLUT isoforms and moderate potency. Biological evaluation demonstrates that these compounds possess clear binding to GLUT4 to impact validated functional end points. Importantly, lead compound 20 was used to

General chemistry experimental

All chemical reagents were obtained from commercial suppliers and used without further purification unless otherwise stated. Anhydrous solvents were purchased from Sigma-Aldrich, and dried over 3 Å molecular sieves when necessary. DCM and THF were purified by passage through a bed of activated alumina. Normal phase flash column chromatography was performed using Biotage KP-Sil 50 μm silica gel columns and ACS grade solvents on a Biotage Isolera flash purification system. Analytical thin layer

Associated content

SMILES table (CSV format) of all final compounds tested in the cell proliferation assay and their proliferation data. 1H and 13C NMR spectra of compound 3, 6, and 20. HRMS-ESI spectrum of compound 20.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from The Searle Funds at The Chicago Community Trust, and Cancer Center Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center and by the American Cancer Society Research scholar grant (

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