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. 2020 Nov 23;3(6):1242–1252. doi: 10.1021/acsptsci.0c00124

The Dihydroorotate Dehydrogenase Inhibitor Brequinar Is Synergistic with ENT1/2 Inhibitors

Christine R Cuthbertson 1, Hui Guo 1, Armita Kyani 1, Joseph T Madak 1, Zahra Arabzada 1, Nouri Neamati 1,*
PMCID: PMC7737209  PMID: 33344900

Abstract

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The dihydroorotate dehydrogenase (DHODH) inhibitor brequinar failed all clinical trials for solid tumors. To investigate mechanisms to increase brequinar’s efficacy, we employed a combination strategy to simultaneously inhibit the nucleotide salvage pathways. Brequinar is synergistic with the equilibrative nucleoside transporter (ENT) inhibitor dipyridamole, but not the concentrative nucleoside transporter inhibitor phlorizin. This synergy carries over to ENT1/2 inhibition, but not ENT4. Our previously described brequinar analogue 41 was also synergistic with dipyridamole as were the FDA-approved DHODH inhibitors leflunomide and teriflunomide but the latter required much higher concentrations than brequinar. Therefore, a combination of brequinar and ENT inhibitors presents a potential anti-cancer strategy in select tumors.

Keywords: DHODH, brequinar, equilibrative nucleoside transporter, dipyridamole, synergy, cancer

To generate the required concentrations of nucleotides for DNA replication and rapid cell growth, cancer cells often use de novo synthesis pathways.1 This differs from normal cells that maintain nucleotide concentrations through nucleoside salvage pathways. This metabolic difference has led to the development of many inhibitors that can selectively target cancer cells by disrupting de novo synthesis.2,3De novo purine biosynthesis inhibitors have become standard-of-care chemotherapies for cancer, and part of their success is due to their transport across the cell membrane by nucleoside transporters.4 5-Fluorouracil (5FU), PALA, acivin, brequinar (BREQ), and leflunomide (LEF)/teriflunomide (TERF) are notable examples of pyrimidine biosynthesis inhibitors. While antimetabolites against purine metabolism have largely been successful, their pyrimidine counterparts have not, with the exception of 5FU, and the reason for this is not clear. LEF/TERF and BREQ all target the enzyme dihydroorotate dehydrogenase (DHODH). LEF, and its active metabolite TERF, are FDA-approved for the treatment of autoimmune diseases such as arthritis and multiple sclerosis.5 Along with autoimmune diseases, DHODH plays a critical role in cancer, which has recently been extensively reviewed.6−9 Despite BREQ’s higher potency and specificity over LEF/TERF (Table S-1), it failed all cancer clinical trials in solid tumors in the late 1980s and early 1990s. We are interested in exploring the potential to improve the effectiveness of the DHODH inhibitor BREQ by identifying synergistic proteins or pathways for a combination therapy.

DHODH is responsible for the oxidation of dihydroorotic acid to orotic acid. This reaction is the fourth, and committed, step in de novo pyrimidine synthesis and its inhibition via BREQ has potent anticancer properties in murine and human xenograft models of cancer.10 Its promising anticancer potential led to multiple clinical trials, but BREQ failed to provide a significant objective response to patients with solid tumors.11−16In vitro, cells grown in nucleoside-supplemented media become insensitive to BREQ,17,18 suggesting a compensatory mechanism exists. A Phase I trial found that after dosing of BREQ, DHODH activity was undetectable in lymphocytes within 15 min and even up to a week afterward,19 but it is not known if tumor samples showed a similar profile. Moreover, as we show and others have seen, continuous treatment of BREQ as a single agent is necessary to observe a cellular effect, so the dosing regimen is also critical.20 Additionally, lack of DHODH activity prevented nearly all tumor growth.21 Clinical trials evaluated efficacy of IV infusions on a bi-weekly basis or for 1 week on, 3 weeks off, so it is unlikely that sustained inhibition was achieved. Furthermore, plasma uridine levels rebounded after a significant initial decrease.19 This suggests that BREQ’s clinical trial failures might have resulted from the salvage pathway importing extracellular nucleosides to sustain cell viability. While the reason that BREQ failed human clinical trials is likely a complicated one, we were interested in gaining further understanding of the role of the salvage transporters as a compensation mechanism as a potential combination strategy.

De novo pyrimidine salvage pathways rely on two families of membrane bound transporters to import extracellular nucleosides: equilibrative nucleoside transporters (ENT1-4/SLC29A1-4) and concentrative nucleoside transporters (CNT1-3/SLC28A1-3).22,23 Since their expression varies with subcellular localization and tissue distribution, normal cells utilize a combination of these two families to import nucleosides as fuel for salvage pathways. However, the ENT is a more effective nucleoside/nucleobase transporter and may present an important synergistic target (ENT1: 200 uridine molecules/s vs CNT1: 10 uridine molecules/s).23,24 The differential expression of the ENTs is cancer-dependent. ENT1 was found to be overexpressed in colon cancer and some pancreatic cancer samples.25−29 There are also numerous reports on the downregulation of ENT1 in pancreatic cancer,30−32 with the majority attributed to a resistance mechanism developed from treatment with purine biosynthesis inhibitors (i.e., gemcitabine).33−46 Therefore, it is possible that these tumors would be more sensitive to pyrimidine biosynthesis inhibition as they would be more reliant on de novo pathways.

The combination of ENT inhibitors with antipyrimidines has been investigated previously in colon and pancreatic cancer including dipyridamole (DPM) + acivicin,47,48 DPM + PALA,49,50 and DPM + PALA + 5FU.51 In fact, in a study exploring the combination of BREQ and 5FU, DPM was found to enhance BREQ’s growth inhibition.20 This was rescued in the presence of 50 μM uridine, but that is well above the estimated physiological concentration of 5 μM,52 which did not rescue the cytotoxicity. In this study, we evaluate synergistic effects of ENT/CNT inhibition concomitantly with DHODH inhibition in colon and pancreatic cancer cells using a small-molecule approach (Figure 1).

Figure 1.

Figure 1

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Structures of inhibitors used in this study. (A) DHODH inhibitors. (B) Transporter inhibitors.

Results and Discussion

Brequinar Is Synergistic with ENT Inhibitor Dipyridamole but Not CNT Inhibitor Phlorizin

We first sought to determine the role of the pyrimidine salvage pathway transporters (CNTs versus ENTs) in the compensatory mechanism for DHODH inhibition. We chose colon and pancreatic cancer cell lines for this study as these are the most well-studied pharmacologically in the context of nucleoside transporters. To accomplish this, we combined BREQ with either phlorizin (PHZ, pan-CNT inhibitor) or dipyridamole (DPM, ENT inhibitor). DPM is selective for ENT1/2, but also inhibits ENT4 at higher concentrations. As single agents, PHZ and DPM are not toxic up to 50 μM (Figure 2A–C). The combination of BREQ + PHZ does not affect the activity of BREQ in the colony formation assay (Figure 2A–C, Figure 2G–I) or MTT assay (Figure S-1A–C) even with continuous and simultaneous treatment. However, treatment of BREQ + DPM for only 24 h is enough to produce significant cytotoxicity (Figure 2A–C). Importantly, this synergy is seen at 100-fold lower concentration of DPM to PHZ. This synergy increases with continuous treatment (Figure 2G–I). The cell lines used in this study show a spectrum of sensitivity to BREQ, with HCT 116 being the most sensitive (Table 1). Even in this cell line, the maximal growth inhibition is roughly 60% (Figure 2D), suggesting that BREQ is a cytostatic drug and that the salvage pathway may sustain ∼40% viability. Regardless of sensitivity to BREQ alone, all of the tested cell lines were sensitive to the combination. A valid concern is that this combination would be toxic to all cells, but, as evidenced by the lack of toxicity in the in vivo study (see below), we believe that with the optimal dosing regimen, systemic toxicity is avoidable. Surprisingly, increasing concentrations of DPM does not affect the IC50 of BREQ, but instead increases the efficacy in a dose-dependent manner (Figure 2D–F), even up to a concentration of 25 μM DPM (Figure S-1D). A similar phenomenon was observed when brequinar was combined with doxorubicin.53 Furthermore, it is interesting to note that neither the order of addition (Figure 2J) nor treatment duration (Figure 2K) significantly affected the IC50 of BREQ. We believe this is due to the intrinsic cytostatic nature of DHODH inhibitors. Inhibition of DHODH results in a stall of cell-cycle progression at S-phase;17,21 cells are able to remain viable because of the salvage pathway. Therefore, when the supply of pyrimidine nucleosides is cut off from both arms, the remaining viable but non-proliferating cells are forced to die (Figure S-1E,F). To validate the cytotoxicity, we carried out MTT assays with compound treatment for only the first 24 h or the full 72 h. Any growth inhibition from the 24 h treatment would be due to cytotoxicity as fresh medium was replaced after compound removal. As can be seen in Figure S-1E, 24 h treatment of BREQ in the MTT assay has a minimal effect, whereas significant growth inhibition is seen with continuous treatment in agreement with BREQ’s cytostatic mechanism of action. Short-term and continuous treatment of BREQ + DPM produces the same effect, indicating a cytotoxic mechanism. Importantly, comparison of both 24 h treatments reveals a significant difference of cell growth inhibition. Additionally, in an apoptosis assay measuring ATP levels, there was not a significant difference in the viability of BREQ-treated cells, signifying that while the cell’s growth was inhibited, the cells were still viable. In contrast, treatment with the BREQ + DPM combination significantly reduced cell viability for 24 h and continuous treatment. These initial studies showed that BREQ is synergistic with DPM but not PHZ, suggesting that CNTs do not play a major role in the salvage pathway when de novo pyrimidine biosynthesis is inhibited in colon and pancreatic cancer cell lines.

Figure 2.

Figure 2

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BREQ is synergistic with ENT antagonist DPM, but not CNT antagonist PHZ. (A–C) Colony formation assay (CFA) at single doses. Continuous and 24 h designations are for combinations only; single drugs are continuous treatments. (D–F) IC50 dependence of BREQ in the presence of DPM in the MTT assay. (G–I) Bliss synergy plots using CFA data. Cells were continuously treated with compounds. (J) Order of addition of BREQ + DPM in the MTT assay in HCT 116. Concentration of DPM = 1 μM. (K) Time-dependence of BREQ + DPM treatment in the MTT assay in HCT 116. BREQ and DPM were added at the same time.

Table 1. IC50 Values of Inhibitors Used in This Study against Cancer Cell Lines. Values Listed Are in μM.

  HCT 116
 HT-29
 MIA PaCa-2
compound MTT CFA MTT CFA MTT CFA
BREQ 0.480 ± 0.14 0.218 ± 0.24 >25 >25 0.680 ± 0.25 0.590 ± 0.36
LEF >50 14.1 ± 2.4 >50 >50 >50 >50
TERF >50 11.8 ± 5.7 >50 >50 >50 23.7 ± 19
41 2.86 ± 0.96 NTa NT NT 7.18 ± 2.3b NT
PHZ >50 >50 >50 >50 >50 >50
DPM >50 >50 >50 >50 >50 >50
8MDP 5.73 ± 2.1 8.30 ± 3.7 >50 >50 16.0 ± 5.0 13.6 ± 6.6
TC-T 6000 11.2 ± 3.9 8.06 ± 3.0 17.9 ± 8.1 6.09 ± 4.0 10.1 ± 2.6 12.0 ± 0.36
NBMPR >50 >50 >50 >50 >50 >50
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a

NT = not tested.

b

Reference #57.

There are a few possibilities as to why we saw synergy between BREQ and DPM but not PHZ. While all CNTs are able to transport uridine, they do so at a less efficient rate than the ENTs.23,24 The transport also requires coupled transport of sodium or protons across their concentration gradient. Additionally, PHZ is not as potent as the ENT inhibitors used in this study. So as to not exceed toxic concentrations of DMSO, we were limited by a maximum concentration of 50 μM. This is 2.5- to 5-fold lower than the IC50 values of CNT1 and CNT2. Therefore, more potent CNT inhibitors such as compound 22 or siRNA for the CNTs could be used in the future to assess whether the combination of DHODH and CNT inhibition is synergistic.54

BREQ Is Synergistic with Other, but Not All, ENT Inhibitors

Beyond DPM, additional ENT inhibitors with varying specificities were tested for synergy with BREQ: TC-T 6000 (ENT4 inhibitor), NMBPR (ENT1 inhibitor), and 8MDP (ENT1/2 inhibitor) (Table S-1). The ENT1/2 specific inhibitors were synergistic with BREQ, whereas the ENT4-specific inhibitor TC-T 6000 was not (Figure 3, Figure S-2). This indicates that ENT4 does not play an important role in salvage of nucleosides in response to DHODH inhibition. All CNTs and ENTs except for ENT4 transport uridine; ENT4 specifically transports adenosine.23 This explains why inhibition by TC-T 6000 did not amount to any synergistic, or even additive, effect. Unlike DPM, 8MDP and TC-T 6000 showed cytotoxicity in all three cell lines (Table 1). This could be due to their higher potency against the ENT isoforms, however, the most selective ENT inhibitor, NBMPR, was not cytotoxic up to 50 μM. It is interesting that these ENT inhibitors show cytotoxicity since not all cell lines are sensitive to BREQ, suggesting that HT-29 and MIA PaCa-2 are more reliant on salvage than de novo pathways. Similar to DPM, 8MDP and NBMPR did not affect BREQ’s IC50, but instead increased its efficacy (Figure 3D,E; Figure S-2C,F). For HCT 116, the synergy is more pronounced with 24 h treatment than continuous (Figure 3G,H) because BREQ also shows growth inhibition with longer treatment times. For MIA PaCa-2, significant synergy is observed with 24 h and continuous treatment, and for HT-29 it is observed only with continuous treatment (Figure S-2). This can be attributed to their intrinsic resistance to BREQ.

Figure 3.

Figure 3

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BREQ is synergistic with ENT1/2 but not ENT4 inhibitors in HCT 116. (A–C) CFA at single doses. Continuous and 24 h designations are for combinations only; single drugs are continuous treatments. (D–F) IC50 dependence of BREQ in the presence of ENT inhibitors in the MTT assay. (G–I) Bliss synergy plots using CFA data.

Other DHODH Inhibitors Show Mixed Synergy with DPM

Additional DHODH inhibitor LEF, and its active metabolite TERF, were also evaluated for synergy with DPM. While only modest cell growth inhibition was observed in the colony formation assay for LEF and TERF alone, combinations with DPM did show a significant improvement (Figure 4A,D; Figure S-3A,D). Both LEF and TERF were inactive in the MTT assay with IC50 values greater than 50 μM (Table 1), but in HCT 116, a combination with DPM increased activity (Figure 4B,E). However, these combinations showed no significant differences in HT-29 or MIA PaCa-2 (Figure S-3B,E). Continuous treatment of LEF/TERF with DPM in the colony formation was synergistic but not with 24 h treatment in HCT 116 (Figure 4C,F). In HT-29 and MIA PaCa-2, there was no observed synergy at concentrations lower than 50 μM for both LEF and TERF (Figure S-3). Since LEF/TERF are minimally active to begin with, when DPM is added an IC50 is produced. In HCT 116, comparison of the Bliss synergy plots for continuous treatments of BREQ + DPM to LEF/TERF + DPM, the trends are quite similar (Figure 1G to Figure 4C,F). By simply looking at the highest concentration of these compounds (25 μM for LEF/TERF or 5 μM BREQ) without DPM, the cell growth inhibition is ∼50–70%. The addition of 0.55 μM DPM increases the cell growth inhibition to greater than 95%, thereby increasing the efficacy without a shift in IC50. This is not apparent in the MTT assay because the cells are only treated with the compounds for 3 days, versus 6 to 7 days in the colony formation assays. LEF and TERF have a much lower affinity for DHODH compared to BREQ (Ki = 4.6 μM and 2.7 μM versus 25 nM, respectively),55,56 which could account for the lack of synergy in HT-29 and MIA PaCa-2 as these cell lines are more resistant to DHODH inhibition.

Figure 4.

Figure 4

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LEF and TERF are synergistic with DPM in HCT 116. (A,D) CFA at single doses. Continuous and 24 h designations are for combinations only; single drugs are continuous treatments. (B,E) IC50 dependence of LEF or TERF in the presence of DPM in the MTT assay. (C,F) Bliss synergy plots using CFA data.

We previously published optimized BREQ analogue 41,57 and were interested whether this molecule would also exhibit synergy when combined with DPM. The cytotoxicity trends with 41 matched those of BREQ + DPM (Figure S-4). Significant synergy is present with both 24 h and continuous treatment (Figure S-4A). Furthermore, DPM dose-dependently increases the efficacy of 41 but does not shift its IC50 value (Figure S-4B). Summaries of all combinations can be found in Tables S-2, S-3, and S-4.

Nucleoside Depletion Confirms Synergistic Mechanism of Action

Removal of nucleosides from the media is a surrogate for inhibition of all nucleoside transporters. Under these conditions, BREQ, TERF, and LEF treatment reproduces the synergy that is seen with combined DPM or NBMPR treatment in nutrient-rich media (Figure 5A). In the MTT assay, removal of nucleosides from the media significantly enhanced activity (Figure 5B). As expected, BREQ was more cytotoxic in nucleoside-free media, and this media did not impact the drug combinations nor the nucleoside transport inhibitors DPM and NBMPR. When uridine was added to the nucleoside-free media (precursor to the end product of pyrimidine biosynthesis) at a physiological concentration (5 μM), BREQ-induced cell growth inhibition was rescued, but not LEF or TERF (Figure 5C). This indicates that the mechanisms of action for LEF and TERF extend beyond DHODH inhibition, and that BREQ’s mechanism of action lies with inhibition of pyrimidine biosynthesis. All together these support previous literature.

Figure 5.

Figure 5

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Nucleoside depletion confirms synergistic mechanism of action. (A) Colony formation assay assessing drug dependence of nucleoside presence in medium. (B) MTT assay assessing drug dependence of nucleoside presence in medium. Drug concentrations are the same as in panel A. *p < 0.05. (C) MTT assay assessing drug dependence of uridine supplementation in dialyzed FBS. Drug concentrations are the same as in panel A. *p < 0.01.

In Vitro Synergy Was Not Observed In Vivo

To evaluate if the in vitro synergy would translate in vivo, the antitumor effects of DPM, BREQ, 41, DPM + BREQ, and DPM + 41 were examined using an HCT 116 mouse xenograft model with i.p. administration (Figure 6A, Figure S-5A). During the study, no significant body weight was lost in any group (Figure 6B, Figure S-5B). While DPM + BREQ showed significant inhibition of tumor growth compared to vehicle control, there was no statistical significance between the combination and BREQ alone (Figure 6C, Figure S-5C). Previous in vivo studies with PALA + DPM found that the combination was able to significantly reduce nucleotide levels in the tumors greater than either agent alone and that addition of DPM reduced PALA’s LD50 in mice.58,59 Therefore, we do not expect tumor penetrance of DPM to be an issue. A possible explanation as to why we did not observe synergy is that plasma uridine concentrations were higher than what was used in cell culture. In mouse plasma the concentration of uridine is ∼5 μM,52 and in Figure 5C, we show that 5 μM does not rescue growth inhibition, therefore we do not expect that plasma uridine levels contributed to the lack of synergy. However, future in vivo studies monitoring plasma uridine levels are warranted as 50 μM was previously shown to rescue the combination effect in vitro,20 and treatment with a subtoxic dose of brequinar in mice resulted in a roughly 3-fold increase in uridine concentrations.19

Figure 6.

Figure 6

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Anticancer effect of DPM + BREQ in a mouse xenograft model (n = 5, mean ± SEM). (A) Formulations of DPM and BREQ. (B) Body weight change throughout the study. (C) Tumor volume of each group. (D) Harvested tumors after sacrifice. *p < 0.001 versus control (t-test).

Disappointingly, 41 showed no antitumor activity alone or in combination with DPM (Figure S-5C). However, as was discussed above, the dosing regimen is important. In this study we only tried one dose combination of BREQ with DPM. In the future it would be important to establish the optimal ratio of DPM/BREQ as well as the schedule-dependency. We were pleased to see that the combination group of DPM + BREQ was not toxic to the mice as measured by body weight. Despite having a favorable pharmacokinetic profile,5741 did not elicit any tumor growth inhibition. In vitro, 41’s IC50 against DHODH is on par with that of BREQ but possesses a roughly 6-fold higher IC50 in cells (HCT 116). Our previous work speculated that even with similar solubility to and a lower cLogP than BREQ, 41 may not be as cell-permeable.57 The computational permeability tool PerMM predicted a 49-fold difference in permeability coefficients between BREQ and 41 (Table S-5), in which BREQ is predicted to more favorably cross the phospholipid bilayer (BLM).60,61 Similarly, the logD7.4 of 41 is more than a full log unit lower than that of BREQ (0.631 ± 0.07 versus 1.83 ± 0.02).62 Moreover, we used a dose of 10 mg/kg for 41 versus 20 mg/kg for BREQ, so it is possible that a higher dose would be more effective. Likewise, the half-life of 41 is ∼2-fold lower than that of BREQ, therefore increasing the dosing schedule to every 12 h instead of daily could potentially overcome the lack of activity. No increased activity was observed in combination with DPM, further indicating a failure to reach the tumor or an inadequate dose. Therefore, future studies are warranted to further elucidate 41’s in vivo efficacy.

Conclusion

This is the first study to thoroughly investigate inhibition of nucleoside transporters in conjunction with DHODH inhibitors. The combination of DPM with BREQ has been studied before, albeit with brevity.20 Through using a small molecule approach, we found that the equilibrative nucleoside transporters seem to play a greater role in nucleotide salvage rather than the concentrative nucleoside transporters when the de novo pathway is inhibited. Experiments using more potent inhibitors of CNTs or siRNA targeting these proteins combined with brequinar are necessary to further validate this result. We identified that the ENT1 and ENT2 isoforms together are responsible for salvage through the use of potent ENT1/2 inhibitors 8MDP and NBMPR. Furthermore, the synergy extended beyond brequinar to other established DHODH inhibitors leflunomide and teriflunomide. The results of our study highlight the possibility of improving brequinar’s efficacy in the clinic by using a combination strategy to inhibit nucleotide salvage from the extracellular environment. Future studies will determine optimal dosing regimens for in vivo studies to obtain a synergistic effect.

Methods

Chemicals

DPM, BREQ, and uridine were purchased from Sigma. Compound 41 was prepared as previously described.57 NBMPR, TC-T 6000, and 8MDP were purchased from Tocris. All stock solutions were prepared in DMSO except for uridine which was dissolved in water. The purity was established by integration of the areas of major peaks detected at 254 nm, and all tested compounds have >95% purity.

Cell Culture

HCT 116 and MIA PaCa-2 were cultured in RPMI 1640, and HT-29 was cultured in McCoy’s 5A; all were supplemented with 10% FBS (Gibco). Dialyzed FBS was purchased from GE Healthcare. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and maintained in culture under 30 passages. Cell lines were tested for Mycoplasma contamination with the Mycoplasma detection kit, PlasmoTest (InvivoGen, San Diego, California). All cell lines were authenticated with STR DNA profiling (University of Michigan, Michigan, USA) and matched to reference profiles from the ATCC database.

Colony Formation Assay

Cells were seeded in 96-well plates at 200–500 cells/well (c/w) and allowed to attach overnight. Compounds were added to the indicated concentrations and incubated with the cells for 24 h or continuously until the vehicle control reached ∼80% confluency (5–7 days). At the end of treatment, media was removed, and cells were fixed and stained with crystal violet solution (0.05% crystal violet, 2% formaldehyde, 40% methanol) for 30 min. Wells were washed with water and allowed to dry overnight before imaging with the Odyssey CLx imager (LI-COR Biosciences).

MTT Assay

The cytotoxicity of the compounds was evaluated with the MTT assay. Cells were seeded in 96-well plates at 2000–4000 c/w. After overnight attachment, compounds were added to the wells at sequential dilutions. After 72, 96, or 120 h the tetrazolium dye MTT was added to the media to a final concentration of 300 μg/mL and incubated for 3–4 h at 37 °C. The media was removed, and the insoluble formazan was dissolved in 100 μL of DMSO. Absorbance at 570 nm was read by microplate reader (Molecular Devices, Sunnyvale, CA). The cytotoxicity of compounds is presented as an inhibition of cell proliferation against DMSO-treated controls. All compound incubation periods were 72 h unless otherwise indicated.

Apoptosis Assay

Apoptosis was measured via quantification of ATP levels using the APOSensor assay (BioVision Incorporated) according to the manufacturer’s protocol. Briefly, HCT 116 cells were seeded in white 96-well plates at 3000 c/w. After overnight attachment, compounds were added to the wells and incubated with the cells continuously or for 24 h. After 72 h the media was removed and 100 μL of nucleotide releasing buffer was added to the wells. ATP monitoring enzyme (10 μL) was added to the wells after the plate was gently shaken for 5 min. Luminescence was read by a CLARIOstar Plus microplate reader (BMG LabTech). Cell viability is presented as a percentage compared to DMSO-treated controls.

Data Analysis

IC50 values were calculated using GraphPad Prism 8. For synergy experiments, data were analyzed for synergistic interactions by the Bliss synergy and antagonism method using the software Combenefit (University of Cambridge).63,64 All experiments were performed at least three independent times.

PerMM Analysis

BREQ and 41 were prepared in MOE using the Amber force field for energy minimizations. Compounds were submitted for analysis by the PerMM server using the default settings (T = 298 K, pH 7.4).60,61

Log D (pH 7.4) Determination

The logD of 41 was first estimated using ADMET Predictor (Simulations Plus, version 9.5.0.16) to inform the appropriate partitions to be used below. The partition coefficient of 41 between n-octanol and PBS at pH 7.4 (log D7.4) was obtained using the shake-flask technique as previously described with some alterations.65 Mutually saturated solutions were prepared by shaking at room temperature overnight. Excess buffer or octanol was removed following centrifugation for 30 min at 3500 rpm. Compound 41 was diluted in 500 μL or 1 mL in octanol-saturated PBS to 200 μM. Aliquots of these standard solutions were transferred to a Corning 3680 plate along with a blank. PBS-saturated octanol was then added to the tubes in 1:1 and 1:10 ratios. The tubes were shaken for 1 h at room temperature and then centrifuged for 5 min at 7000 rpm. Aliquots of the aqueous layers were transferred to the plate, and the absorption spectra for all samples were obtained. The area under the curves (AUC) was calculated in Microsoft Excel. Equation 1 was used to determine the log D and the reported value is an average of six experiments.

graphic file with name pt0c00124_m001.jpg 1

Xenograft Study

All animal studies were approved by the animal care facility at the University of Michigan-Ann Arbor (Protocol number: PRO00009185) and were handled in accordance with the Institutional Animal Use and Care Committee. HCT 116 cells (5 × 106) in a suspension of PBS were injected subcutaneously into dorsal flanks of NSG mice. Tumor size was monitored twice a week through caliper measurement using the following equation: V = l × w × w/2, where l represents length and w represents width of the tumor. Mice were randomly grouped (n = 5 per group) when the average tumor size reached 150 mm3. Daily treatment was given at 5 days on, 2 days off cycles. DPM was given at 10 mg/kg, compound 41 at 10 mg/kg, and BREQ was given at 20 mg/kg (10% DMSO, 50% PEG400, 40% saline) by intraperitoneal injection. Study was concluded on day 25 when the average tumor size in the vehicle-treated group reached 2000 mm3. An unpaired t-test was performed for data analysis, and p < 0.05 was considered significant.

Acknowledgments

C.R.C. is a trainee of the University of Michigan Pharmacological Sciences Training Program (PSTP, T32-GM007767). This work was supported by NIH Grant R01 CA188252 and a grant from the University of Michigan Forbes Institute for Cancer Discovery. We thank Dr. Essam Eldin A. Osman for assistance with spectral and purity analysis, and Dr. Osman and Maha Hanafi for assistance with computational predictions. We also thank Dr. Andrea Shergalis for critical reading of the manuscript.

Glossary

Abbreviations

BREQ

brequinar

LEF

leflunomide

TERF

teriflunomide

PHZ

phlorizin

DPM

dipyridamole

DHODH

dihydroorotate dehydrogenase

ENT

equilibrative nucleoside transporter

CNT

concentrative nucleoside transporter

5FU

5-Fluorouracil

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00124.

  • Additional figures as described in the text; table summarizing biochemical and clinical characteristics of drugs used in this study; tables summarizing synergy results; table summarizing output from PerMM calculations (PDF)

Author Contributions

C.C., H.G., A.K., Z.A., and J.T.M. performed the experiments. C.C., H.G., A.K., and Z.A. analyzed and interpreted the data. C.C., J.T.M., and N.N. wrote the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt0c00124_si_001.pdf (740.3KB, pdf)

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