Main Research Findings

1) Deazaflavin and related analogues were found to inhibit tyrosyl DNA phosphodiesterase 2, as well as sensitize cancer cells towards treatment with topoisomerase II poison etoposide.

2) The study highlighted the development of specific deazaflavin TDP2 inhibitors through the selection of drugs that specifically target etoposide in TDP2-proficient cells.

 

Selected Data

1) This study performed by the researchers Kankanala et al focused on the rational design, synthesis, and characterization of novel deazaflavin analogues as potent inhibitors of tyrosyl DNA phosphodiesterase 2 (TDP2) and their ability to sensitize cancer cells to etoposide (ETP) treatment. The research involved a multifaceted approach combining medicinal chemistry, biochemical assays, cell-based studies, and computational molecular modeling [1].

The chemical synthesis of the deazaflavin scaffold, specifically analogues of subtype 4, followed previously established procedures. This involved a sequential reaction beginning with 6-chlorouracil and substituted amines to yield the 6-amino intermediate. Subsequent condensation of 15 with 4-cyano-2-fluorobenzaldehyde furnished the core deazaflavin structure. To create the novel subtype 11 analogues, a key synthetic strategy involved the preparation of specific amine intermediates. Intermediates 20 were synthesized from N-Boc protected 3- or 4-aminophenol by treating it with substituted benzyl bromides to form O-benzyl derivatives, followed by N-Boc deprotection to yield the aniline derivatives. Alternatively, amine intermediate 21 was synthesized through a reaction of phenolic alcohol 22 with 4-nitrophenyl fluoride 23, followed by nitro group reduction [1].

These amine intermediates were then reacted with 6-chlorouracil to form intermediate 25, which upon condensation with 4-cyano-2-fluorobenzaldehyde, produced the desired subtype 11 analogues. For the N-3 substituted deazaflavin subtype 12, a modified Chan-Lam coupling was employed, reacting scaffold 4 directly with substituted boronic acids under copper(II) acetate catalysis. An alternative linear synthesis for subtype 12, particularly for analogues 12b, 12j-k, and 31, was also developed, starting with the synthesis of 1-phenylpyrimidine-trione from urea 27 and diethylmalonate, followed by chlorination and amination reactions. All compounds were rigorously characterized using ¹H and ¹³C NMR spectroscopy, high-resolution mass spectrometry (HRMS-ESI), and their purity (≥ 97%) confirmed by HPLC with detection at 254 nm.

The biological evaluation commenced with assessing TDP2 inhibitory activity using a fluorescence-based biochemical assay. Compounds were tested in a dose-response fashion, and IC50 values were determined from three independent experiments performed in triplicate. The assay utilized a reaction buffer composed of Tris-HCl, MgCl2, KCl, Tween-20, and DTT, with 14M_TDP2 enzyme and a fluorescently labeled substrate. Membrane permeability, a critical factor for cellular potency, was evaluated using the Parallel Artificial Membrane Permeation Assay (PAMPA). This assay involved a 96-well filter plate pre-coated with lipids, with compound solutions of 10 µM in DPBS, added to donor wells and DPBS to receiver wells. After a 5-hour incubation at 25 °C, the concentrations in both wells were analyzed by LC-MS/MS, and effective permeability coefficients (Pe) were calculated [1].

Cytotoxicity of the synthesized analogues was assessed in two human cancer cell lines: HepG2 (human liver carcinoma), commonly used for HBV infection studies, and HeLa (human cervical adenocarcinoma), permissible to picornavirus infection. Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, following a 72-hour incubation with serially diluted compounds. CC50 values were calculated from data fitted in GraphPad Prism software. The sensitizing effect of the novel inhibitors on cancer cells was evaluated in DT40 chicken lymphoma cells, a well-established model for TDP2 inhibition studies. DT40 cells were treated with combinations of ETP and the TDP2 inhibitors, and cell viability was measured after 72 hours using the ATPlite luminescence assay. The decrease in cell viability with increasing inhibitor concentrations at a constant ETP dose was interpreted as a cooperative interaction indicating sensitization.

Finally, molecular modeling and docking analyses were performed using Schrodinger’s small molecule drug discovery suite. The co-crystal structure of the humanized mouse TDP2 catalytic domain bound to compound 4c was retrieved from the protein data bank. This structure was prepared and refined using Maestro and protein preparation wizard tools. Ligand preparation and docking were conducted using LigPrep and Glide XP, respectively, after defining the active site around the native ligand, 4c. The docking poses were further refined by post-docking minimization. This computational approach aimed to elucidate the binding modes of the novel analogues within the TDP2 active site and understand structure-activity relationships (SAR) by identifying critical molecular interactions, including hydrogen bonds and cation-π interactions [1].

2) This comprehensive study completed by the research team of Kiselev et al investigated novel deazaflavin analogues, ZW-1231 and ZW-1288, as potent inhibitors of tyrosyl DNA phosphodiesterase 2 (TDP2) and explored their potential to sensitize cancer cells to etoposide (ETP) and mitoxantrone. The research involved a multi-faceted approach, encompassing chemical synthesis, biochemical assays, cell-based studies, and molecular modeling, all conducted with a comparative focus on a previously established deazaflavin inhibitor, SV-5-153 [2].

The study initiated with the synthesis of the deazaflavin compounds. The base deazaflavin scaffold was derived from 6-chlorouracil through a series of reactions involving substituted amines and condensation with 4-cyano-2-fluorobenzaldehyde. The novel analogues, ZW-1231 and ZW-1288, were obtained by replacing the tetrazole moiety of SV-5-153 with a p-hydroxy group, followed by further derivatization. ZW-1231 featured a p-hydroxy modification, while ZW-1288 involved an N-3 substituted aromatic ring. Details of the synthesis for the base SV-5-153 and its modifications to yield ZW-1231 and ZW-1288 were reported in a preceding publication. All chemical structures were confirmed, and compounds were deemed pure for biological testing.

Biochemical assessment of TDP2 inhibitory activity was performed using a recombinant human TDP2 (hTDP2) assay. The reactions were carried out using a 32P-labeled DNA oligonucleotide substrate (TY18) bearing a 5′-phosphotyrosyl group, incubated with recombinant hTDP2 in the presence or absence of inhibitors. The enzymatic cleavage reactions were performed in a Tris-HCl buffer supplemented with KCl, MgCl2, EDTA, DTT, BSA, and Tween 20. Reactions were terminated, and products were separated by denaturing PAGE, followed by phosphorimaging and densitometry analysis to determine IC50 values. To evaluate the specificity of the inhibitors, their activity against recombinant human TDP1 was also tested using a 5′-[32P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y) substrate. The cross-resistance profile was investigated using a hTDP2 L313H single-point mutant and mouse TDP2 (mTDP2), for which previous studies showed resistance to SV-5-153. These experiments aimed to discern if the novel analogues maintained the selective mechanism of inhibition observed with the parent compound [2].

To bridge the gap between biochemical and cellular activity, a whole cell extract (WCE) TDP2 assay was conducted. This involved collecting, lysing, and centrifuging hTDP2-complemented DT40 knockout cells (TDP2-/-). The WCEs were then incubated with the TY18 DNA substrate in the presence or absence of inhibitors, and TDP2 cleavage activity was measured as described for the recombinant assay. This assay provided insight into the drug’s activity in a more complex cellular environment, accounting for potential non-specific binding or drug sequestration.

Cell-based studies were crucial to evaluate the therapeutic potential of the inhibitors. Cytotoxicity as a single agent was assessed in three cancer cell lines: chicken lymphoblast DT40, human prostate DU145, and human leukemia CCRF-CEM. Cells were seeded into 96-well plates and continuously exposed to various concentrations of the drugs for 72 hours. Cell viability was then determined using the ATPlite luminescence assay, measuring ATP levels as an indicator of viability. Synergism with TOP2 poisons was evaluated by treating cells with combinations of ETP or mitoxantrone and the deazaflavin inhibitors. The dose-response curves were analyzed using CompuSyn software to calculate Combination Index (CI) values, where CI values less than 1 indicated synergism. The selectivity of this sensitizing effect was further tested by combining ZW-1288 with the TOP1 inhibitor camptothecin and the PARP inhibitor olaparib. To explore the underlying mechanisms of synergy, intracellular ETP levels were measured in DT40 cells treated with ETP alone or in combination with SV-5-153 or ZW-1288. Cells were counted using a cell viability analyzer, lysed, and intracellular ETP levels quantified using LC/MS/MS [2].

Finally, molecular modeling provided insights into the binding modes of SV-5-153, ZW-1231, and ZW-1288 with hTDP2. The hTDP2 model was constructed using SWISS-MODEL with mTDP2 structure as a template. The mTDP2 crystal structure was trimmed, and the protein structure was geometry-optimized. Ligand structures were prepared and optimized. Docking was performed, covering the catalytic site and DNA binding channel. This computational approach aimed to elucidate the specific molecular interactions contributing to inhibitor potency and selectivity [2].

 

Discussion

1) The investigation performed by the research team of Kankanala et al into novel deazaflavin analogues yielded significant results regarding their biochemical potency, membrane permeability, cytotoxicity, and their ability to sensitize cancer cells to etoposide (ETP) treatment, offering promising candidates for TDP2 inhibition [1].

The initial evaluation of a control series centered around analogue 4c (a subtype 4 deazaflavin) confirmed previous findings, showing potent TDP2 inhibition in the nanomolar range. However, this series, particularly analogues 4a-c with polar substituents, exhibited extremely low PAMPA permeability measured by Pe in the range of 0.003-0.01 × 10⁻⁶ cm/s, indicating poor cellular uptake. While removal or etherification of the hydroxyl group (4d, 4e-h) improved permeability substantially, it often led to a decrease in inhibitory activity. Notably, analogue 4f, designed for balanced potency and permeability, showed moderate potency at concentrations of 0.88 µM and improved permeability compared to highly polar analogues [1].

The study then focused on two novel subtypes: subtype 11, introducing an additional phenyl ring off ring A, and subtype 12, with a phenyl ring at the N-3 site of the deazaflavin scaffold. For subtype 11 with an O-substituted phenyl ring A, prominent SAR trends revealed that benzyl ethers generally exhibited higher inhibitory potency than phenyl ethers. Specifically, compounds 11a and 11e showed comparable potency to 4c but drastically improved permeability by 75-150 fold, demonstrating a successful balance of properties. Analogues 11 l-m, based on the meta phenolic analogue 4b, also showed strong TDP2 inhibition and improved permeability, albeit with higher IC50 values than 4b.

The N-3 arylated subtype 12 analogues consistently showed improved TDP2 inhibition compared to subtype 11. Most subtype 12 compounds, particularly those with para-phenolic alcohol (12a-f) or meta-phenolic alcohol (12g-i) on ring A, displayed 2-6 fold improved potency over 4c or 4b. For instance, analogue 12g demonstrated the highest potency observed to date in the study, representing a substantial improvement over previous deazaflavin inhibitors. Moderate improvements in permeability by 3-13 fold were also observed across subtype 12 analogues, with the exception of 12f. Further modification of N-10 phenyl A substituents in subtype 12 revealed that removing the polar phenolic OH group resulted in compound 12l with much reduced inhibitory activity but improved permeability. Analogues 12j-k, incorporating an NHSO₂Me group, achieved single-digit nanomolar IC50 values, marking them as the most active inhibitors tested, despite their lower permeability. Overall, the N-3 arylation proved to be a highly productive modification, leading to a new deazaflavin subtype with enhanced TDP2 inhibition and PAMPA permeability [1].

Cytotoxicity evaluation in HepG2 and HeLa cells indicated a strong correlation between cytotoxicity and PAMPA permeability. For subtypes 4 and 11, compounds with low permeability generally showed no significant cytotoxicity up to 100 µM, while those with higher permeability tended to be more cytotoxic. Notably, analogue 12l from subtype 12 exhibited high permeability and cytotoxicity, while some other subtype 12 analogues displayed cytotoxicity despite lower permeability, attributed to their extraordinary TDP2 inhibitory potency.

Crucially, the novel inhibitors demonstrated strong sensitizing effects on DT40 chicken lymphoma cells toward ETP treatment. Compounds 11a, 11e, 12a, 12b, and 12h consistently showed much stronger cancer cell sensitizing effects compared to the previously reported best analogue, 4a. Even compounds like 12h, which showed some cytotoxicity at higher concentrations of 25 µM, reached a plateau in enhancing ETP action at concentrations below 10 µM, indicating a cooperative interaction. This extraordinary sensitizing effect is attributed to a balanced profile of TDP2 inhibition and PAMPA permeability for these novel analogues [1].

Molecular modeling provided crucial insights into the binding modes of these new subtypes. Building upon the known binding of 4c, which involves hydrogen bonding with R266/R268 and N264 via a water molecule, the novel designs aimed to utilize empty spaces within the binding pocket. Subtype 11, incorporating an O-substituted phenyl ring, and subtype 12, with an N-3 substituted phenyl ring, were successfully docked. Representative analogues 11k and 12b interacted extensively with R266 and R268. Specifically, the ether linker of 11k allowed its phenyl substituent to engage with H226, N264, and H349. Uniquely, subtype 12’s N-3 aryl group oriented parallel to R268, enabling cation interactions, which molecular modeling and SAR observations strongly suggested contributed to the improved potency observed in this subtype. The enhanced binding due to these cation-π interactions between the N-3 aryl and the R268 residue of TDP2 explained the superior biochemical activity of subtype 12 analogues. The overall profiles validate these novel deazaflavin analogues as effective cellular probes with improved properties over prior inhibitors [1].

2) The study completed by researchers Kiselev et al successfully characterized novel deazaflavin analogues, ZW-1231 and ZW-1288, demonstrating their potent TDP2 inhibitory activity and strong cancer cell sensitizing effects, while elucidating key aspects of their mechanism of action.

Initially, biochemical evaluation of recombinant hTDP2 inhibition confirmed the nanomolar potency of all tested deazaflavins. SV-5-153 exhibited an IC50 of 81 nM, while ZW-1231 showed 275 nM, and ZW-1288 demonstrated 102 nM. These results indicated that the chemical modifications introduced in ZW-1231 and ZW-1288 maintained the high inhibitory potency characteristic of the deazaflavin scaffold against hTDP2. Crucially, the study assessed the cross-resistance profile of these compounds against hTDP2 L313H mutant and mTDP2. All three compounds showed lower efficiency against hTDP2 L313H, with IC50 values ranging from 4.6 µM to 5.8 µM, representing a 17- to 64-fold resistance compared to wild-type hTDP2. Moreover, none of the deazaflavins effectively inhibited mTDP2 at concentrations up to 111 µM, confirming a common and selective mechanism of inhibition, consistent with previous findings on SV-5-153 [2].

Further supporting their cellular relevance, the deazaflavins retained their inhibitory potency in WCE TDP2 assays. SV-5-153, ZW-1231, and ZW-1288 inhibited TDP2 in WCE with IC50 values of 28 nM, 89 nM, and 96 nM, respectively. The slight enhancement in potency in cellular extracts suggested minimal drug sequestration or non-specific binding to other cellular proteins, underscoring their robust activity in a more physiological context. Importantly, all three compounds demonstrated high selectivity, showing no detectable activity against recombinant human TDP1, thus confirming their specific targeting of TDP2 [2].

 

Figure 1: Changes in inhibitory potency in the presence of deazaflavin analogues, SV-5-153, ZW-1231, and ZW-1288.

In cell-based studies, ZW-1288 displayed limited single-agent cytotoxicity across the three tested cancer cell lines: chicken lymphoblast DT40, human prostate DU145, and human leukemia CCRF-CEM. Its 50% growth inhibition (GI50) values were 49.4 µM in DT40, 22.2 µM in CCRF-CEM, and 52.9 µM in DU145, comparable to the parent compound SV-5-153 with measurements of GI50 >100 µM in DT40, 24.3 µM in CCRF-CEM, and 85.7 µM in DU145. In contrast, ZW-1231 showed higher cytotoxicity as a single agent measuring at GI50 6.5 µM in DT40, 6.1 µM in CCRF-CEM, and 8.4 µM in DU145, suggesting potential off-target effects beyond TDP2. This favorable toxicity profile made ZW-1288 an ideal candidate for further drug combination studies.

A pivotal finding was ZW-1288’s strong synergistic activity with the TOP2 poison ETP. In DT40 cells, ZW-1288 synergized with ETP, significantly enhancing its cytotoxic action even at concentrations where ZW-1288 alone exerted no toxicity. This synergy was maintained even in DT40 TDP2-knockout cells and in Mre11-deficient cells, indicating that ZW-1288 possesses additional cellular targets or mechanisms beyond solely inhibiting TDP2 or Mre11. The dose-response curves for ETP/ZW-1288 combinations showed a steeper incline compared to ETP/SV-5-153, with CI values for ETP/ZW-1288 combinations consistently indicating strong synergism. This synergistic effect was selective for TOP2 poisons, as ZW-1288 did not amplify the cytotoxicity of the TOP1 inhibitor camptothecin or the PARP inhibitor olaparib. Furthermore, ZW-1288 also synergized with mitoxantrone in CCRF-CEM and DU145 cells, demonstrating its broad applicability with TOP2 poisons across various cancer types [2].

Investigation into intracellular ETP levels revealed that both SV-5-153 and ZW-1288 increased ETP uptake in DT40 cells, with ZW-1288 showing a greater effect. While SV-5-153 at 3 µM increased intracellular ETP 8-fold with only ~10% increase in cell death, ZW-1288 at 1 µM resulted in a more modest ETP increase but a much stronger cytotoxic effect. This suggested that the observed synergy is not solely attributable to enhanced ETP accumulation but likely involves additional, more complex intracellular activities of the deazaflavins.

Molecular docking analyses provided structural insights into the binding modes. SV-5-153, ZW-1231, and ZW-1288 were found to bind within the hTDP2 DNA binding channel. The binding mode of SV-5-153 showed placement perpendicular to the DNA binding channel, forming hydrophobic contacts with L313 and polar contacts with R366. ZW-1231 exhibited a similar binding mode, with its introduced benzyl group fully occupying the L313 surface area. ZW-1288, however, displayed a novel binding pose with its fused tricyclic motif aligned along the DNA binding channel, and the N-3 phenyl substituent interacting with L313. These docking results were consistent with biochemical findings regarding the role of L313 in inhibitor performance and cross-resistance, highlighting the impact of structural modifications on binding affinity and cellular efficacy [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citation

[1] Kankanala J, Ribeiro CJA, Kiselev E, et al. Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide. J Med Chem. 2019;62(9):4669-4682. doi:10.1021/acs.jmedchem.9b00274

[2] Kiselev E, Ravji A, Kankanala J, Xie J, Wang Z, Pommier Y. Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors. DNA Repair (Amst). 2020;85:102747. doi:10.1016/j.dnarep.2019.102747

 

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