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An Enzyme-Directed Imidazoquinoline Activated by Drug Resistance Anthony J. Burt, Joseph D. Hantho, Amy E. Nielsen, and Rock J. Mancini Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00095 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018
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Biochemistry
An Enzyme-Directed Imidazoquinoline Activated by Drug Resistance Anthony J. Burt, Joseph D. Hantho, Amy E. Nielsen *, and Rock J. Mancini * Department of Chemistry, Washington State University, 100 Dairy Road, Pullman, WA 99164
Supporting Information Placeholder ABSTRACT: Drug efflux and enzymatic drug degradation are two cellular mechanisms that contribute to drug resistance in many cancers. Herein, we report the synthesis and in vitro activity of a pro-immunostimulant that exploits both processes in tandem to selectively confer cancer-mediated immunogenicity. We demonstrate that an imidazoquinoline pro-immunostimulant is inactive until it is selectively metabolized to active immunostimulant by endogenous α-mannosidase enzyme expressed within multidrug-resistant cancer cells. Following conversion, the immunostimulant is transported to the extracellular space via drug efflux, resulting in the activation of model bystander immune cells. Taken together, these results suggest that enzyme-directed immunostimulants can couple immunogenicity to these mechanisms of drug resistance. We name this process BystanderAssisted ImmunoTherapy (BAIT), and envision that it could be advanced to treat drug-resistant diseases that rely on enzymatic degradation or drug efflux to persist.
MultiDrug-Resistant (MDR) cancers evade the action of chemotherapeutics through two prevalent mechanisms: drug efflux and enzymatic drug degradation.1,2 Transport proteins from the ATP Binding Cassette superfamily are implicated in drug efflux.3 Of these, P-Glycoprotein (P-gp) is the most well-studied transport protein that participates in drug efflux.4,5 P-gp is highly promiscuous. Diverse substrates are all susceptible to P-gpmediated drug efflux,6,7 with no single correlation in substrate specificity other than a weak association with hydrophobicity.8 This promiscuity is particularly problematic for chemotherapeutic drugs. P-gp in MDR cancers causes efflux of chemotherapeutics from the cytosol to the extracellular space, resulting in decreased intracellular drug concentration and diminished efficacy.9 Directed Enzyme Prodrug Therapy (DEPT) attempts to outcompete drug efflux by increasing the local concentration of therapeutic drug within the tumor microenvironment. In DEPT, enzymes within the tumor microenvironment convert an enzymedirected prodrug to an active therapeutic drug.10–12 Several examples of DEPT are established for both exogenous and endogenous enzyme systems,13,14 with DEPT strategies often including selfimmolative spacers that optimize enzymatic conversion of prodrug to active drug.15 However, one aspect common to all DEPT approaches is the potential for diffusion of active drug away from the target cell following enzymatic conversion (Figure 1a). This results in off-target activity termed the bystander effect.16 The bystander effect can be beneficial, resulting in the destruction of nearby cancer cells,17,18 or undesirable, resulting in systemic toxicity from off-target effects.19 Regardless, a common paradigm is that MDR cancers exhibit drug efflux.20 This enhances bystander effects in MDR cancers. In this work, we examined replacing the chemotherapeutic typically used in DEPT with an immunostimulant. In vitro, we demonstrate that this modification makes the bystander effect a desirable outcome, causing MDR cancer cells to elicit activation of model bystander immune cells.
Figure 1. a) Previous Work: Directed Enzymatic Prodrug Therapy (DEPT), features an enzyme substrate and self-immolative spacer covalently attached to a chemotherapeutic. In the treatment of MDR cancers, drug efflux causes off-target bystander effects. b) This Work: An example of Bystander-Assisted ImmunoThapy (BAIT). When MDR cancers are treated with α-mannosidasedirected pro-immunostimulants, the resistance mechanisms of enzymatic conversion and drug efflux are exploited in tandem, resulting in the activation of bystander immune cells. In both cases, cellular uptake, enzymatic conversion, and drug efflux occur. Because this novel mechanism of action relies on MDR cancermediated conversion of pro-immunostimulant, followed by the activation of bystander immune cells, we have named our approach Bystander-Assisted ImmunoTherapy (BAIT). In BAIT, a pro-immunostimulant is first converted to active immunostimulant by enzymes within MDR cancer cells. The liberated immunostimulant then co-opts drug efflux, via P-gp or comparable transport proteins, resulting in transport to the extracellular space. This allows the immunostimulant to activate bystander immune cells, resulting in immunogenicity mediated by MDR cancer cells (Figure 1b). To rationally design a pro-immunostimulant that would be amenable to BAIT, we drew inspiration from our previous report of the first enzyme-directed imidazoquinoline immunostimulant.21 We hypothesized that this enzyme-directing approach could be used to create an α-mannosidase-directed pro-immunostimulant whereby α-mannosidase, upregulated in a variety of cancers,22 could be exploited to drive conversion of pro-immunostimulant to active immunostimulant, followed by efflux to the extracellular space. For our immunostimulant we chose Imiquimod, a representative of the imidazoquinoline family of immunostimulants that is known to confer anti-cancer immunogenicity when applied directly to solid tumors.23,24 Currently, Imiquimod is limited to topical applications due to the inflammatory toxicity that results
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from systemic routes of administration.25–28 However, imidazoquinoline activity can be modulated by modification of the aminoquinoline moiety.29–32 This provides an ideal location for attachment of a mannopyranoside-directing group that will target the pro-immunostimulant to α-mannosidase upregulated in cancer cell metabolism. To test our hypothesis that an α-mannosidase-directed imidazoquinoline would be amenable to BAIT, we chose the AT3B-1 MDR prostate cancer cell line. AT3B-1 cells were chosen because they upregulate P-gp in response to epigenetic pressure of the chemotherapeutic doxorubicin included in their media formulation.33 We suspected AT3B-1 cells would additionally have upregulated α-mannosidase because overexpression of the gene MAN2C1, encoding for α-mannosidase, contributes to tumorigenesis in MDR prostate cancers.34 To test this, α-mannosidase in the AT3B-1 cell line was quantified by measuring cell-mediated conversion of 4-nitrophenyl-α-mannopyranoside (α-mannosidase substrate). This resulted in α-mannosidase activity of 88 ± 14 pU cell-1, a 3-fold increase in enzyme relative to immune cells (Figure S7). Based upon these findings, we rationally designed our proimmunostimulant to create an imidazoquinoline mannopyranoside that was predicted to possess α-mannosidase-dependent immunogenicity. This was achieved by covalent attachment of an αmannosidase-cleavable group and self-immolative spacer to the parent immunostimulant Imiquimod (Figure 2a). Briefly, synthesis of the pro-immunostimulant began with D-(+)-mannose (1). Sequential peracetylation, bromination, and Koenigs-Knorr substitution with 4-hydroxy-3-nitrobenzaldehyde provided the Oglycoside (2) in 47% combined yield over 3 steps. Next, (2) was reduced to alcohol (3) by treatment with sodium borohydride, followed by formation of the activated carbonate (4) via addition to p-nitrophenyl chloroformate. Carbamoylation with the immunostimulant Imiquimod to form (5) was achieved using a microwave reactor. The protected pro-immunostimulant (5) was then subjected to Zemplén deacetylation conditions to provide the imidazoquinoline-mannopyranoside (6) in 3% total linear yield from D-(+)-mannose over 7 steps (For complete synthetic details, see Supporting Information).
Figure 2. a) Synthetic route to pro-immunostimulant (6); a: i) Ac2O, pyridine, DCM, rt, 2 h; ii) HBr 33% (w/w) in AcOH, DCM, rt, 3 h; iii) 4-hydroxy-3-nitrobenzaldehyde, Ag2O, ACN, rt, 4 h; b: NaBH4, MeOH 25% (v/v) in DCM, rt, 10 min; c: 4nitrophenyl chloroformate, pyridine, DCM, reflux, 24 h; d: Imiquimod, DIPEA, EtOAc, Microwave (90 W), 87 °C, 45 min; e: NaOMe, MeOH, 0 °C, 1 h. b) Conversion of proimmunostimulant (6) to Imiquimod (m/z = 597.80 and 241.10 Da, respectively) by exogenous α-mannosidase or AT3B-1 cells was monitored by LCMS. c) Kinetics of conversion for 10 µM (6) driven by 0.1 U mL-1 exogenous α-mannosidase (Blue), 2.5 x 106 cells mL-1 AT3B-1 multidrug-resistant prostate cancer cells (Red), or cell media (Grey). The t1/2 of conversion was determined to be 3 h and 19 h for α-mannosidase and AT3B-1 cells, respectively. Error bars represent 1 standard deviation from the mean of experiments repeated in triplicate: * p
