Research paperDevelopment of GLUT4-selective antagonists for multiple myeloma therapy
Graphical abstract
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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