High expression of class III β-tubulin has no impact on functional cancer cell growth inhibition of a series of key vinblastine analogs

Abstract

Clinical association studies have implicated high expression of class III β-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III β-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3–10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III β-tubulin unlike the taxanes. Most significant are the results with 4–6, a subset of 20′ amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2–2 fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux.

Graphical Abstract

The Vinca alkaloids are a family of natural products that continue to have a remarkable impact on anticancer drug discovery and treatment.^1,2 Originally isolated in trace quantities from the periwinkle plant (Catharanthus roseus (L.) G.Don),^3,4 vinblastine (1) and vincristine (2) are the most prominent members of this class and among the first plant-derived natural products used in the clinic for the treatment of cancer (Figure 1). These two compounds along with three clinically-approved semi-synthetic analogs, vindesine,⁵ vinorelbine⁶ and vinflunine,⁷ are integral oncology drugs employed today in highly successful combination drug therapies. Their mode of action, which involves disruption of microtubulin formation and dynamics through tubulin binding, remains one of the most successful approaches for the discovery and development of new oncology drugs.⁸

Figure 1.

Structures of vinblastine and vincristine.

Although vinblastine and vincristine are superb drugs even by today’s standards, a potential limitation to their continued use is the emergence of clinical resistance. Most recognized of the mechanisms of resistance to the Vinca alkaloids is that mediated by overexpression of the drug efflux pump phosphoglycoprotein (Pgp).⁹ Pgp overexpression, which also results in multidrug resistance (MDR), is responsible for the majority of all relapses in oncology. The discovery of vinblastine analogs not susceptible to Pgp efflux could serve as potential replacements for vinblastine in its current clinical uses or in instances of Pgp-derived vinblastine resistance. Even more significantly, it could expand the use of a vinblastine to new therapeutic applications for Pgp-derived MDR tumor treatments. Despite past efforts focused on vinblastine that have searched for analogs that effectively overcome Pgp-derived vinblastine resistance, little progress has been made.² Recent advances in the total synthesis of vinblastine, vincristine and related natural products provided access to analogs of the natural products not previously accessible by semisynthetic modification of the natural products themselves.^10–12 The latest of these efforts provided a powerful approach to access vinblastine analogs that contain systematic deep-seated modifications within either the lower vindoline-derived^13–21 or upper catharanthine-derived subunits.^22–29 As a result of these developments, we have disclosed several series of key analogs, systematically exploring and defining the impact individual structural features and substituents have on tubulin binding affinity and cancer cell growth inhibition. In these studies, we have shown that replacement of the C20′-OH with 20′ ureas was possible,²² that substantial^23,24 and even remarkable²⁵ potency enhancements were obtainable with such 20′ ureas, and that some exhibited further improvements in activity against vinblastine-resistant cancer cell lines.²⁴ In an extension of these studies, an extensive series of vinblastine 20′ amides were disclosed that provided analogs that matched or exceeded the potency of vinblastine, but that are not subject to Pgp efflux and its derived vinblastine resistance.²⁶ Although not discussed herein, well-defined structure–activity results and accompanying structural models were delineated that account for not only the on target tubulin binding affinity and resulting functional cell growth inhibition of the analogs (HCT116), but also defined the structural features and their characteristics needed for avoidance of Pgp efflux and resistance derived from Pgp overexpression (HCT116/VM46). The studies provided vinblastine analogs no longer susceptible to resistance derived from overexpression of Pgp, and they represented the discovery of a site and functionalization strategy for the preparation of readily accessible vinblastine analogs (3 steps) that improve binding affinity to tubulin (on target affinity) and functional potency in cell-based assays while simultaneously disrupting their efflux by Pgp (off target affinity and source of resistance), offering a powerful opportunity to discover new, improved, and durable oncology drugs.

Alterations to the target tubulin could also impact activity and contribute to or be responsible for Vinca alkaloid resistance. A series of association studies of clinical data have implicated high level expression of class III β-tubulin as both a prognostic and predictive factor for lower response rates or reduced overall survival in patients receiving tubulin binding drugs.^30,31 However, most of the association studies and the supporting cellular studies have examined the impact of class III β-tubulin on taxanes and a much smaller sampling of its impact on Vinca alkaloids are represented in the association studies.^30,31 Despite the obvious differences in the tubulin binding sites of the taxanes and Vinca alkaloids as well as their distinct functional behaviors (stabilization vs destabilization of tubulin dynamics), both taxanes and the Vinca alkaloids typically have been lumped together as potentially being negatively impacted by the high expression of class III β-tubulin.^30,31 Because of these implications, we have examined a series of the key vinblastine analogs that emerged from our studies in additional cell-based functional cell growth inhibition assays for their sensitivity to the high expression of class III β-tubulin and report the results herein.

The key analogs that were examined alongside vinblastine (1) are 10′-fluorovinblastine (3),²⁸ three vinblastine 20′ amides 4–6 that displayed no susceptibility to Pgp efflux and are insensitive to Pgp overexpression resistance,²⁶ and a series of vinblastine 20′ ureas 7–10 that includes the ultrapotent analogs 8–10 (Figure 2).^24,25 In our original work, these were screened for growth inhibition activity against HCT116 (human colon cancer cell line) and a matched resistant cell line (HCT116/VM46) that is approximately 100-fold resistant by virtue of the overexpression of Pgp. This well-designed set of cell-based assays simultaneously provided a direct measure of both functional activity (HCT116) and the analog susceptibility to Pgp efflux (resistance, HCT116/VM46). Key members were assessed for tubulin binding affinity for correlation with functional activity and those that showed no sensitivity to Pgp overexpression, including 4–6, were examined in efflux assays to confirm their behavior toward Pgp and related efflux transporters.²⁶ The cell growth inhibition activity against the L1210 (mouse leukemia) cell line was also measured and the results were qualitatively and quantitatively (IC₅₀) nearly identical to those observed with the HCT116 cell line. In these studies, the HCT116 human tumor cell line was found to accurately reflect activity observed against a larger panel of clinically relevant human tumor-derived cell lines.^12,24–28 Alongside this prior data and herein, we now report the cell growth inhibition activity of the analogs 3–10 against the human A549 cell line (human non-small cell lung cancer) and a matched cell line A549-T24 insensitive to taxol (Figure 2).³² After its original generation and characterization, this taxol-resistant cell line (A549-T24) was found to embody an increased expression of class III β-tubulin that was correlated with the loss in taxol sensitivity with no alteration in Pgp expression.³² As a result, the comparison of cell growth inhibition of candidate drugs against A549 vs A549-T24 has been used to characterize potential resistance due to increased expression of class III β-tubulin.³²

Figure 2.

Cell growth inhibition of A549 versus A549-T24 cancer cell growth summarized alongside previous reported data for L1210, HCT116 and HCT116/VM46. All compounds were >95% pure and have been previously disclosed (3,²⁸ 4–6,²⁶ 7,²⁴ and 8–10²⁵).

Consistent with prior reports, A549-T24 proved insensitive to taxol treatment, exhibiting a 10-fold loss in potency relative to wild type A549 cells (IC₅₀ = 70 vs 7.1 nM).³² In contrast, vinblastine as well as all the vinblastine analogs 3–10 did not exhibit any loss in sensitivity toward A549-T24. In fact, most showed slight increases in potency (1–3.6 fold, avg = 1.8 fold), suggesting they may be even more effective in the presence of expressed class III β-tubulin. In retrospect, this may not be surprising. Class III β-tubulin increases the instability and dynamics of microtubule assembly, potentially countering the stabilizing effects of bound taxol, but enhancing the destabilizing effects of the Vinca alkaloids. Most significant within this series of analogs are the results with 4–6.²⁶ They match or exceed the potency of vinblastine, they display equipotent or more potent activity against A549-T24 than even wild type A549 cells (1.2–2 fold) and, as detailed earlier,²⁶ they uniquely display no sensitivity to the overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to efflux by Pgp or related drug efflux transporters.²⁶ The results not only suggest that (1) vinblastine and related analogs are not likely to be prone to resistance derived from high expression of class III β-tubulin unlike the taxanes, but also that (2) association studies of clinical data with tubulin binding drugs^30,31 should treat taxanes and the Vinca alkaloids as distinct drug classes likely to exhibit different sources of on target resistance.