Bifunctional Duocarmycin Analogues as Inhibitors of Protein

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2019, 82, 16−26

Bifunctional Duocarmycin Analogues as Inhibitors of Protein Tyrosine Kinases Christian De Ford,†,‡,§ Kamala Penchalaiah,⊥,§ Alexander Kreft,⊥ Matjaz Humar,† Wolfgang Heydenreuter,∥ Mehrnoush Kangani,⊥ Stephan A. Sieber,∥ Lutz F. Tietze,*,⊥ and Irmgard Merfort*,†,‡

J. Nat. Prod. 2019.82:16-26. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/25/19. For personal use only.

†

Department of Pharmaceutical Biology and Biotechnology, Albert Ludwigs University Freiburg, Stefan-Meier-Strasse 19, D-79104 Freiburg, Germany ‡ Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Albertstrasse 19a, 79104 Freiburg, Germany ⊥ Institute of Organic and Biomolecular Chemistry, Georg-August University, Tammannstrasse 2, 37077 Göttingen, Germany ∥ Institute of Organic Chemistry II, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany S Supporting Information *

ABSTRACT: Bifunctional duocarmycin analogues are highly cytotoxic compounds that have been shown to be irreversible aldehyde dehydrogenase 1 inhibitors. Interestingly, cells with low aldehyde dehydrogenase 1 expression are also sensitive to bifunctional duocarmycin analogues, suggesting the existence of another target. Through in silico approaches, including principal component analysis, structure-similarity search, and docking calculations, protein tyrosine kinases, and especially the vascular endothelial growth factor receptor 2 (VEGFR-2), were predicted as targets of bifunctional duocarmycin analogues. Biochemical validation was performed in vitro, confirming the in silico results. Structural optimization was performed to mainly target VEGFR-2, but not aldehyde dehydrogenase 1. The optimized bifunctional duocarmycin analogue was synthesized. In vitro assays revealed this bifunctional duocarmycin analogue as a strong inhibitor of VEGFR-2, with low residual aldehyde dehydrogenase 1 activity. Altogether, studies revealed bifunctional duocarmycin analogues as a new class of naturally derived compounds that express a very high cytotoxicity to cancer cells overexpressing aldehyde dehydrogenase 1 as well as VEGFR-2.

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This prompted us to search for new targets that might be involved in the cytotoxic mechanism of bifunctional duocarmycin analogues. In silico approaches revealed tyrosine kinases as potential targets, which were validated by in vitro assays using the bifunctional analogue 2. Furthermore, a new bifunctional duocarmycin analogue, 10, without alkylating properties was predicted, synthesized, and confirmed to inhibit VEGFR-2. Bifunctional duocarmycin analogue 10 is only a weak inhibitor of aldehyde dehydrogenase 1 in contrast to 2. Cell-based assays indicated that those cancer lines known to overexpress VEGFR-2 as well as aldehyde dehydrogenase 1 were strongly influenced by 2, but to a much lesser degree by 10.

uocarmycin SA (1) is an antibiotic metabolite isolated from Streptomyces DO-113 that exerts very high cytotoxic1 and antitumoral effects2 in the picomolar range. This natural compound acts through sequence-specific DNA alkylation and interstrand cross-linking of AT-rich regions of the minor groove.3 However, SA 1 and its analogues so far failed as cancer chemotherapeutics, as significant hematologic toxicity has been observed in clinical trials.4 To circumvent such undesired side effects, different derivatives were synthesized, such as the bifunctional duocarmycin analogues5,6 with an IC50 = 150 fM that lack the indole moiety as DNAbinding unit (e.g., 2). These compounds lost the DNA alkylating properties, but instead, they were shown to be irreversible inhibitors of aldehyde dehydrogenase 1.5−7 However, bifunctional duocarmycin analogues also exhibit strong cytotoxic effects in cells with a low expression of this enzyme.8 Therefore, another target could be responsible for the high cytotoxicity of bifunctional duocarmycin analogues in those cells.7,9 © 2019 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In Silico Approaches Reveal Tyrosine Kinases as a Further Target for Bifunctional Duocarmycin AnaReceived: March 20, 2018 Published: January 8, 2019 16

DOI: 10.1021/acs.jnatprod.8b00233 J. Nat. Prod. 2019, 82, 16−26

Journal of Natural Products

Article

between two molecules according to their steric and electrostatic features to find a consensus 3D arrangement. Several VEGFR-2 inhibitors were shown to be among the top results (Figure S2a,b). Hence, three different chemometric approaches were used to find possible new targets for the bifunctional duocarmycin analogues. Bifunctional duocarmycin analogue 2 showed similarities to several tyrosine kinase inhibitors depending on the analysis used. Therefore, a correlation of all the approaches was performed, and the most similar compounds were obtained by Euclidean and Manhattan distance calculation. Altogether, imatinib, cabozantinib, and masitinib exhibited the highest structural similarity to 2 (Figures 2d, S3), suggesting ABL-2, VEGFR2, and c-KIT as possible targets. Most tyrosine kinase inhibitors can be classified as either type I or type II inhibitors, depending on their binding interactions with the kinase domain.27 Kinase inhibitors that bind in the Asp-Phe-Gly (DFG)-out or inactive conformation are called type II inhibitors, whereas if they bind in the DFG-in or active conformation, they are called type I inhibitors. All the tyrosine kinase inhibitors obtained in the in silico analysis as structurally related to bifunctional duocarmycin analogue 2 are classified as type II inhibitors (Figure S4). According to our in silico results, 2 could also target ABL-2, VEGFRs, and c-KIT in a type II inhibition mode.23,28,29 To test this hypothesis, we generated a model of the inhibitory complexes by molecular modeling using GOLD 5.2. To determine if GOLD 5.2 is able to reproduce the cocrystal structures, the ligands of each protein were removed from the binding site and redocked, and the root-mean-square deviation (RMSD) was calculated. GOLD 5.2 was proven suitable to reproduce the crystal structures showing RMSD values below 1 Å (Figure S5 and Table S3). The kinase domain is made up of two lobes connected by the hinge region allowing the lobes to rotate upon ATP and substrate binding; the ATP-binding site is located in a pocket between the two lobes.30 The DFG motif, which is the beginning of the activation loop, guides the transition between the inactive and the active conformations of the tyrosine kinases (TKs) by rotating the Asp residue. Therefore, compounds that interact with the hinge region and the DFG motif (such as imatinib, cabozantinib, and masitinib) potently block the kinase activity.30 The docking analyses gave high scores for bifunctional duocarmycin analogue 2 using the tyrosine kinases ABL-2, VEGFR-2, and c-KIT and very similar docking scores to motesanib and VEGFR-2 (Table 1). The binding mode showed that 2 might be a type II inhibitor, corroborating our hypothesis. Thus, the linker of the CBI subunits of 2 laid above the DFG motif, thereby presumably preventing the conformational change of the activation loop and consequently the kinase activity. Interestingly, 2 was predicted to create a hydrogen bond with the DFG motif of VEGFR-2 but only hydrophobic interactions with ABL-2 and c-KIT. On the other hand, π−π interactions and hydrogen bonds are predicted between the hinge region of ABL-2 (Figure 3a) and VEGFR-2 (Figure 3b) and one of the CBI subunits of 2, in the same way as imatinib and motesanib, respectively. Control docking experiments with another VEGFR-2 crystal with axitinib as cocrystal tyrosine kinase inhibitor (PDB: 4AG8) and with a cKIT-sunitinib complex (PDB: 3G0E) showed the same results (data not shown). In addition, docking experiments were also carried out with VEGFR-1 and VEGFR-3 (Figures S6 and S7)

logues. At first a principal component analysis (PCA) with ChemGPS-NP10 was carried out to explore the chemical space of bifunctional duocarmycin analogues and to compare them with a recently published database of chemotherapeutic compounds with defined mechanisms of action.11 ChemGPSNP analyzes the physicochemical properties of molecules by extracting 35 molecular descriptors subdivided in eight principal components (PC).12 PC1−4 are responsible for 77% of the data variance; therefore, the data were analyzed as previously described.10,11,13 The PCA clustered the bifunctional duocarmycin analogues within the chemical space of the protein tyrosine kinase inhibitors (Figure 1a,b). Moreover, the Euclidean distance between 2 and all the tyrosine kinase inhibitors was calculated using PC1−4 values. Bifunctional duocarmycin analogue 2 was found to be close to imatinib and ponatinib (ABL-2, KIT inhibitors),14,15 cabozantinib (VEGFR2, KIT inhibitor),16 masitinib (KIT inhibitor),17 and lapatinib (EGFR inhibitor),18 all of which are marketed drugs used in the clinic for the treatment of cancer (Figure 1c,d). A second in silico approach was conducted to study in more depth the structural similarity of bifunctional duocarmycin analogues to chemotherapeutic compounds from the used database and to all drugs deposited in the Drugbank.19 For that task, a similarity search approach using Indigo fingerprints (IF; Indigo Cheminformatics toolkit) to calculate the Tanimoto similarity coefficient (TSC) was carried out by constructing a data pipeline in Knime (Figure S1, Supporting Information) and setting a similarity cutoff value at 0.5. IF is a topological path-based fingerprint consisting of up to 2048 bits and encoding all possible connectivity pathways through a molecule; the same principle is used by Daylight fingerprints.20 TSC was chosen as an in silico tool because it is the best method for computation of fingerprint-based similarity.21,22 It became obvious that the bifunctional duocarmycin analogue 2 shows a remarkable resemblance to multiple tyrosine kinase inhibitors (Figure 2a). Interestingly, 2 displayed a similar molecular fingerprint to motesanib, cabozantinib (Figure 1d), and sunitinib, potent vascular endothelial growth factor receptor-2 (VEGFR-2), and KIT inhibitors (Figure 2b),16,23−25 and to SU4984 (Figure 2c), an experimental inhibitor of the fibroblast growth factor receptor 1 (FGFR1),26 respectively. Table S2 presents all the compounds used for the analysis. In addition, an overlay similarity coefficient (OSC) calculation was carried out with Discovery Studio 4.0 to compare the 3D arrangement of the molecular structures of tyrosine kinase inhibitors and 2. OSC measures the overlap 17



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Figure 1. In silico analysis of bifunctional duocarmycin analogue 2. Chemographic mapping of the chemical space of 2 displaying PC1−3 (a) and PC2−4 (b). PC1 describes size, shape, and polarizability; PC2, aromatic and conjugation properties; PC3, H-bond capacity, polarity, and lipophilicity; and PC4, structural flexibility and rigidity. Chemotherapeutics with the same mode of action are presented with the same color. AM: antimetabolites, AA: alkylating agents, T1: topoisomerase I inhibitors, T2: topoisomerase II inhibitors, AT: antitubulin agents, KI: tyrosine kinase inhibitors, CS: cardiotonic steroids, SP: SERCA pump inhibitors. Bifunctional duocarmycin analogues are shown as yellow dots. (c) Euclidean distance (ED) of 2 to all tyrosine kinase inhibitors of the database. A cutoff value of 1.5 was set to show the closest compounds for further analysis. (d) The five closest compounds to 2 in the tyrosine kinase inhibitors cluster according to the ED calculations and 2. All bifunctional duocarmycin analogues used in the analysis are shown in Table S1.

to determine if 2 may be able to preferentially inhibit isoform 2, the most important isoform that mediates angiogenesis.31 The results predicted 2 to bind strongly to isoform 2, followed by isoform 1, and to show less affinity to isoform 3 (Table 1). On the other hand, although GOLD predicted good docking scores for 2 in the ATP-binding site of c-KIT, the crucial interactions needed to potently inhibit its activity are not present (Figure 3c). So far we have focused our studies on the in silico prediction of a novel target for bifunctional

duocarmycin analogues lacking a DNA-binding moiety and demonstrated through chemometric and modeling approaches that 2 could be a novel type II tyrosine kinase inhibitor. Bifunctional Duocarmycin Analogue 2 Differently Inhibits Tyrosine Kinases. To confirm our in silico results, in vitro inhibitory effects of 2 against the same five tyrosine kinases were studied. The phosphorylation activity of tyrosine kinases was measured using a radiometric assay (33PanQinase activity assay) and a range of concentrations of 2 (1−10 000 18



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Figure 2. Tanimoto similarity coefficient (TSC) analysis and in silico correlations. (a) TSC values of bifunctional duocarmycin analogue 2 in descending order with all the compounds from our chemotherapeutic compound database. (b) Structures of the compounds with TSC values over the cutoff value; for the structure of cabozantinib see Figure 1d. (c) Most similar compound to 2 from the Drugbank.19 (d) Correlation of the three in silico approaches. The Euclidean and Manhattan distances were calculated using the values obtained in each approach (TSC, OSC, and ChemGPS-NP) and compared to find the most similar compounds to 2. The top three compounds are shown with arrows. All the distances can be found in Figure S3. OSC: overlay similarity coefficient. ED ChemGPS-NP: Euclidean distance obtained using PC1−4 values from the PCA with ChemGPS-NP.

Table 1. Effects of Bifunctional Duocarmycin Analogue 2 on Tyrosine Kinase Inhibition in Silico and in Vitro tyrosine kinase ABL-2 c-KIT VEGFR-1 VEGFR-2 VEGFR-3

docking score 2 46.91 44.37 39.84 44.10 36.40

± ± ± ± ±

0.92 1.79 1.83 1.18 1.48

docking score KIa 48.99 49.41 45.38 45.20 38.29

± ± ± ± ±

1.31 1.09 0.41 0.17 0.71e

ΔGb/kcal mol−1

IC50/nM 685 3000 1275 530 4200

± ± ± ± ±

320 200 425 150 1000

−8.5 −7.6 −8.1 −8.6 −7.4

± ± ± ± ±

0.3 0.1 0.2 0.2 0.1

c TK/nM

equivc

LEd/kcal mol−1 non-H−1

1.5 12.9 4.5 2.3 4.7

456.7 232.6 283.3 230.4 893.6

−0.218 −0.195 −0.208 −0.221 −0.190

a

Scores of the redocked crystal structures. bExperimental free energy of binding. cEquivalents of compound necessary to inhibit 50% of the enzymatic activity. dLigand efficiency is the energy of binding per non-hydrogen atom of an inhibitor; BDA 2 has 39 non-H atoms. eVEGFR-3 is a homology model predicted with Phyre2; thus motesanib was docked to obtain a docking score.

of both subunits and no modification on the linker could enhance tyrosine kinase inhibition, as shown for the bifunctional duocarmycin analogue 10 in Table S5. Moreover, any group at R1 capable of forming hydrogen bonds may decrease the affinity to both tyrosine kinases. The introduction of nitrogen on the linker (R2) also considerably decreased the docking scores. A recent report showed that bifunctional duocarmycin analogues with nitrogen in the linker present higher IC50 values in cancer cells in comparison to the analogues with an sp3 carbon at the same position,6 being in line with the decrease of the docking scores. Docking experiments with duocarmycin resulted in low docking scores, indicating that this compound does not fit well in the binding site of these two kinases due to the presence of a DNA-binding unit. Figures 5 and S8 show the interactions of bifunctional duocarmycin analogue 10 in the binding site of VEGFR-2 and

nM). Bifunctional duocarmycin analogue 2 inhibited the tyrosine kinases in a concentration-dependent manner. VEGFR-2 was the most sensitive tyrosine kinase followed by ABL-2. Bifunctional duocarmycin analogue 2 showed the best ligand efficiencies for these tyrosine kinases (Table 1). Higher concentrations even led to a complete inhibition of the phosphorylation activity (Figure 4). Optimization of Bifunctional Duocarmycin Analogues for Tyrosine Kinase Inhibition by Docking Experiments and Validation by Biochemical Testing. Bifunctional duocarmycin analogue 2 was shown to strongly inhibit VEGFR-2 and ABL-2. For structure optimization toward both tyrosine kinases slight structural modifications of the bifunctional duocarmycin analogue backbone and the linker were carried out, and the new molecules were docked. Modification at R1 predicted that removal of the chlorine atom 19



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Figure 3. Docking models with GOLD 5.2. Prediction of the binding mode of bifunctional duocarmycin analogue 2 in the ATP binding site of ABL-2 (a), VEGFR-2 (b), and c-KIT (c). The hinge region is shown in green and the DFG motif in light blue. Orange lines represent π interactions, green lines denote hydrogen bonds, and blue lines denote nonclassical hydrogen bonds.

duocarmycin analogue 10 was selected as the most promising compound. For the synthesis of bifunctional duocarmycin analogue 10 compound 3 was treated with 1,1′-thiocarbonyldiimidazole to give 4, which reacted with tris(trimethylsilyl)silane, leading to the CBI unit 5 with a methyl instead of a chloromethyl group at C-1 in an excellent overall yield. 5 could further be transformed to 5a, which however could not be deprotected. Therefore, the debenzylation was performed with 5 to furnish 6, which after acetylation gave 7. Finally, N-Boc deprotection using HCl and reaction with glutaryl chloride provided 8 and 9, which were deprotected to give the desired bifunctional duocarmycin analogue 10. Deacetylation could also be performed with pure 8 with slightly higher yield (see Scheme 1 and Supporting Information). To corroborate the in silico predictions, phosphorylation activity using the same five tyrosine kinases as employed with bifunctional duocarmycin analogue 2 were tested in the presence of increasing concentrations of 10, showing the strongest inhibition of the activity of VEGFR-2 in vitro (Table 2 and Figure 4).

ABL-2. The binding modes of 2 and 10 are nearly identical; however, the methyl group of 10 was predicted to be in the same position as one of the methyl groups of motesanib in the binding site, partially explaining the increase in the predicted activity (scores) compared to 2 (Figure S8f). Addition of a methyl group often makes a molecule more hydrophobic and more likely to bind to biomolecules. However, the placement of a methyl group in a hydrophobic environment must be in the correct position to show such effects.32 In addition, the removal of the chlorine facilitates 10 to better interact with the DFG motif of VEGFR-2 (Figure 5b). Bifunctional duocarmycin analogues with chlorine at R1 can undergo Winstein cyclization to form the corresponding spirocyclopropylcyclohexadienone moiety. This structural feature has been shown to be essential for the irreversible inhibition of aldehyde dehydrogenase 1 via a nucleophilic attack and adduct formation with Cys302.9 Removal of the chlorine atom would render a bifunctional duocarmycin analogue unable to undergo Winstein cyclization, and no covalent adduct would be formed, switching the mechanism of action most probably to tyrosine kinase inhibition. Based on the docking scores for VEGFR and ABL-2 bifunctional 20



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Figure 4. Effects of bifunctional duocarmycin analogues 2 and 10 on tyrosine kinase phosphorylation activity. Data represent means ± SD of two independent experiments. A: activity.

Figure 5. Docking results of bifunctional duocarmycin analogue 10 in the catalytic domain of ABL-2 (a) and VEGFR-2 (b). Orange lines show π interactions, green lines denote hydrogen bonds, and blue lines show nonclassic hydrogen bonds.

Biological Evaluation of Bifunctional Duocarmycin Analogues 2 and 10. It is known that tyrosine kinases are overexpressed by many tumor types and their expression correlates with clinical parameters.31 Lung and breast cancers, as well as acute lymphoblastic leukemia (T-ALL), have been shown to overexpress VEGFRs to promote vascularization of the primary tumor or enhance aggressiveness.33,34 In this regard, targeting the VEGFR-2 has been considered to be an efficient route to develop antitumor agents.18,23 Recently, the effect of bifunctional duocarmycin analogues on the viability of cancer cell lines was reported, showing strong cytotoxic effects in cells with high as well as low aldehyde dehydrogenase 1 expression.8 Continuing these studies, we included three TALL cell lines (CCRF-CEM, CEM-ADR5000, and Jurkat) overexpressing VEGFRs35 and aldehyde dehydrogenase 1,36 a

pancreatic carcinoma (MIA-PaCa-2) overexpressing aldehyde dehydrogenase 137 but lacking VEGFRs,38 and the already tested non-small-cell lung cancer cell line A549 overexpressing both aldehyde dehydrogenase 18 and VEGFRs39 in our experiments. T-ALL cell lines CCRF-CEM and CEMADR5000 were also recently shown to be cytotoxically affected by artesunic acid homo- and heterodimers40 as well as thymoquinone−artemisinin hybrids.41 Using the MTT assay bifunctional duocarmycin analogue 2 strongly decreased cell viability in cancer cells, especially in cells overexpressing both aldehyde dehydrogenase 1 and VEGFRs, whereas in MIA-PaCa-2 cells lacking VEGFRs reduction of cell viability was lower (Table 3, Figure S9). These findings correspond with those obtained by Tercel et al.,8 who showed that 2 is cytotoxic in breast cancer cells 21



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Scheme 1. Synthesis of Bifunctional Duocarmycin Analogue 10

Table 2. Effects of Bifunctional Duocarmycin Analogue 10 on Tyrosine Kinase Inhibition in Vitro tyrosine kinase ABL-2 c-KIT VEGFR-1 VEGFR-2 VEGFR-3

IC50/nM 540 8300 2100 330 >10 000

± ± ± ±

64 2300 280 28

ΔGa/kcal mol−1 −8.6 −7.0 −7.8 −8.9

± ± ± ±

c TK/nM

equivb

LEc/kcal mol−1 non-H−1

1.5 12.9 4.5 2.3 4.7

360 643 511 143

−0.233 −0.189 −0.209 −0.240

0.05 0.1 0.1 0.04

a

Experimental free energy of binding. bEquivalents of compound necessary to inhibit 50% of the enzymatic activity. cLigand efficiency is the energy of binding per non-hydrogen atom of an inhibitor; bifunctional duocarmycin analogue 10 has 37 non-H atoms.

Table 3. Effects of Bifunctional Duocarmycin Analogues 2 and 10 on the Cell Viability of Cancer Cells and Normal Cells from Healthy Humans Using the MTT Assay 2 a

cells

PBMC CCRF-CEM CEM-ADR5000 Jurkat A549 MIA-PaCa-2

type of cells

IC50 (nM)

normal cells T-ALL T-ALL T-ALL NSCLC pancreatic carcinoma

± ± ± ± ± ±

2000 0.9 6.9 1.4 2.7 14.1

400 0.3 2.1 0.3 0.2 0.3

10 SI

b

IC50 (nM)

SIb

c

2222 290 1429 741 142

>80 000 182 ± 57 >10 000 660 ± 240 663 ± 360 >10 000

>439 >121 >120

a PBMC: peripheral blood mononuclear cells. T-ALL: T-cell acute lymphoblastic leukemia. NSCLC: non-small-cell lung cancer. bSelectivity index, IC50 PBMC cells/IC50 cancer cells. cDetermination of IC50 was not possible, as 80 μM decreased viability only to 80%. The compounds were incubated for 24 h with the cells at 37 °C and 5% CO2.

to those cells that overexpress both proteins. Thus, in the presence of both targets, a stronger effect on cell viability is observed, whereas if one of them is lacking, the activity

(MDA-231 and MCF-7 cells) with low expression of aldehyde dehydrogenase 1 but overexpressing VEGFRs.42 Interestingly, the effect on cell viability is reduced by a factor of 10 compared 22



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Figure 6. Effect of bifunctional duocarmycin analogues 2 and 10 on the cell cycle of cancer cells. (a) Representative histograms of cancer cells treated with bifunctional duocarmycin analogues or axitinib. (b) Cell cycle distribution of cancer cells treated with bifunctional duocarmycin analogues for 24 h. Increase in the sub-G1 population indicates apoptosis. Axitinib was used as a positive control of VEGFR-2 inhibition.

conditions as recently published for 2.6 Whereas an IC50 of 0.46 μM was reported for 2 (obtained in time-dependent inhibition experiments for this irreversible inhibitor), we determined an IC50 of 5.6 μM for 10 (Figure S10). Although 10 cannot covalently bind to aldehyde dehydrogenase 1 as reported for 2,6 interactions between the aromatic residues of aldehyde dehydrogenase 1 and 10 may result in a reversible inhibition of this enzyme. However, this inhibitory effect on aldehyde dehydrogenase 1 may be low compared to the effect on VEGFR-2 and may explain the different effect of 2 and 10 on the investigated cancer cell lines. Interestingly, axitinib, a potent VEGFR-2 inhibitor,44 showed a similar behavior to 10 in the cancer cell lines tested. To further investigate the effects of bifunctional duocarmycin analogues in cancer cells, the cell cycle distribution was evaluated using flow cytometry analysis after 24 h of incubation. As shown by the representative histograms (Figure 6a), significant alterations of the cell cycle of cancer cells were observed in response to bifunctional duocarmycin analogues. Bifunctional duocarmycin analogue 2 preferentially induced apoptosis in a concentration-dependent manner, as observed by the increase in sub-G1 population in the range of 1 μM to

decreases. Recently, VEGF signaling through VEGFR-2 was reported to increase the abundance of cancer cells with aldehyde dehydrogenase 1 activity, which is a stem-cell-like feature.43 Therefore, the potent effect on cell viability of 2 may be explained by a dual synergistic inhibition of both aldehyde dehydrogenase 1 and VEGFR-2. On the contrary, peripheral blood mononuclear cells (PBMC) from healthy human donors were considerably less affected by 2, highlighting the potential of these bifunctional duocarmycin analogues as promising future cancer chemotherapeutics. The effect on cell viability of bifunctional duocarmycin analogue 10 was also tested in the same cancer cell line panel. IC50 values considerably increased compared to 2 in those cell lines overexpressing both aldehyde dehydrogenase 1 and VEGFR-2 (Table 2, Figure S9), but they are still in the nanomolar range. Interestingly, 10 was unable to reduce the cell viability up to 10 μM in MIA-PaCa-2 cells, which only overexpress aldehyde dehydrogenase 1. Cell viability of PBMCs was not affected by 10. To confirm that the different impact on cell viability of 2 and 10 in these cancer cell lines may be dependent on aldehyde dehydrogenase 1, 10 was studied for its inhibitory activity on this enzyme using the same 23



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drugbank.ca/). The best matches were consequently analyzed to obtain further information on the mechanism of action. An arbitrary cutoff value of 0.5 was set to obtain the most similar compounds. Overlay Similarity Coefficient with Discovery Studio 4.0. The energy of the compounds was minimized using the MM2 protocol of ChemBio3D Ultra 13.0 and saved as mol files, which were exported to Discovery Studio 4.0. Rotational bonds were added, and a flexible alignment was carried out finding a consensus between both molecules. The overlay similarity coefficient considers steric and electrostatic features of the compounds. Thus, positioning of functional groups in the same volume contributes to a higher OSC. An arbitrary cutoff value of 0.75 was set to obtain the most similar compounds. Docking Calculations with GOLD 5.2. The crystal structures of VEGFR-1 (PDB: 3HNG), VEGFR-2 (PDB: 3EFL), ABL-2 (PDB: 3GVU), and c-KIT (PDB: 1T46)28 were downloaded from the protein data bank50 and subjected to docking studies using GOLD 5.2 software (CCDC, Cambridge, UK). A homology model of the VEGFR-3 kinase domain was created using the Phyre2 protein fold recognition server51 (http://www.sbg.bio.ic.ac.uk/phyre2/) since no crystal structure has been reported to date. The protein sequence (accession number NP_891555.2) was obtained from the national center for biotechnology information (NCBI, http://www.ncbi.nlm. nih.gov/protein/) and submitted in FASTA format to Phyre2; a cut off of 40% sequence homology was set to obtain better results. Docking experiments were executed three times using the default docking settings with a total of 30 genetic algorithm (GA) runs for each compound. To accelerate the calculations, the program was allowed to stop the GA runs when the top three solutions were within 1.5 Å RMSD. The active site radius was set at a distance of 13 Å from the aspartic acid of the DFG motif present in each active site. Intermolecular interactions were described using Discovery Studio 4.0 (Accelrys Inc., San Diego, CA, USA). Protein Kinase Assay. A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of the five protein kinases. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, MA, USA) in a 50 μL reaction volume. The reaction cocktail was pipetted in four steps in the following order: 10 μL of nonradioactive ATP solution (in H2O), 25 μL of assay buffer/[γ-33P]-ATP mixture, 5 μL of test sample in 10% DMSO, 10 μL of enzyme/substrate mixture. The assay for all protein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, ATP (variable amounts, corresponding to the apparent ATP-Km of the respective kinase, Table S4), [γ-33P]-ATP (approximately 1.7 × 106 cpm per well), protein kinase (variable amounts; Table 1), and substrate (variable amounts; Table S4). All protein kinases provided by ProQinase (Freiburg, Germany) were expressed in Sf9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins, either as full-length or enzymatically active fragments. All kinases were produced from human cDNAs. Kinases were purified by affinity chromatography using either GSH-agarose or immobilized metal. Affinity tags were removed from a number of kinases during purification. The purity of the protein kinases was examined by SDSPAGE/Coomassie staining. The identity of the protein kinases was checked by mass spectroscopy. The concentrations of enzymes and substrates used for the assays are shown in Table S4. The reaction cocktails were incubated at 30 °C for 60 min. The reaction was stopped with 50 μL of 2% (v/v) H3PO4, and plates were aspirated and washed two times with 200 μL 0.9% (w/v) NaCl. Kinase activity dependent transfer of 33Pi (counting of “cpm”) was determined with a microplate scintillation counter (Microbeta, Wallac). All assays were performed with a BeckmanCoulter Biomek 2000/SL robotic system. Aldehyde Dehydrogenase 1 Activity Assay. To ensure formation of equilibrium, different concentrations of bifunctional duocarmycin analogue 10 (1 μL of DMSO stock) were incubated in Tris-HCl 50 mM (46.5 μL) and 2.5 μL of aldehyde dehydrogenase 1 (2.5 μL, 8.2 μM, 410 nM final concentration) for 2 h at 30 °C. Subsequently, a 50 μL substrate mixture containing 50 mM Tris-HCl, 100 mM KCl, 5 mM β-mercaptoethanol, 1 mM β-NAD+, and 10 mM

10 nM (Figure 6b). However, at 1 nM, 2 induced strong S arrest in all the cell lines tested except in MIA-PaCa-2 cells. On the contrary, 10 induced accumulation of cells in the S phase of the cell cycle at 1 μM and promoted strong G2/M arrest at lower concentrations (100−1 nM) in most of the cell lines tested. In a similar way to 2, 10 was unable to induce cell cycle arrest in MIA-PaCa-2 cells, which lack VEGFRs.38 These results suggest that the presence of VEGFRs is necessary for the induction of cell cycle arrest by bifunctional duocarmycin analogues. A similar profile of cell cycle arrest was observed with 100 nM axitinib. These results are in line with the cell viability assays. Axitinib has been shown to induce strong cell cycle arrest in cancer cells, especially at the G2/M phase in a cell-dependent manner.45 In conclusion, using in silico approaches, we found protein tyrosine kinases as new targets for the naturally derived bifunctional duocarmycin analogues that lack a DNA-binding subunit. Depending on the substitution, bifunctional duocarmycin analogues of both types, 2 and 10, may be interesting therapeutic options in cancer therapy. Bifunctional duocarmycin analogues like 2 may be effective against cancer cells overexpressing both aldehyde dehydrogenase 1 and VEGFR. Here, antibody-directed enzyme prodrug therapy (ADEPT), to selectively activate the compounds in cancer cells, may be a suitable strategy, as it has been previously reported with bifunctional duocarmycin analogues.46−48

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EXPERIMENTAL SECTION

General Experimental Procedures. Unless stated otherwise, the reactions were performed under an argon atmosphere in flame-dried flasks, and the reactants were introduced by syringe or transfer cannula with pressure using argon. All solvents were reagent grade and stored over molecular sieves. All reagents obtained from commercial sources were used without further purification. Longterm cooling was performed by using the cryostat EK 90 from the Haake company. Thin-layer chromatography was carried on precoated silica gel plates Si 60 F254 from Merck (Darmstadt, Germany). Silica gel 60 (0.040 0.063 mm) from Merck was used for flash column chromatography. Staining was accomplished using phosphomolybdic acid hydrate (in MeOH) from Sigma-Aldrich (Darmstadt, Germany). Yields refer to isolated and purified compounds, unless stated otherwise. NMR spectra were recorded on a Varian Mercury-300, Unity-300, and Inova-600 spectrometer and a Bruker AMX-300 spectrometer in CDCl3, CD3OD, or d6-DMSO. ESIMS and ESIHRMS spectra were recorded on a Bruker Daltonik Apex IV, IR spectra on a Bruker Vector 22 spectrometer, and UV spectra on a JASCO V-630 spectrometer. Principal Component Analysis of Duocarmycin Analogues with ChemGPS-NP and Cluster Separation by CheS-Mapper. The chemical space of duocarmycin analogues was explored with ChemGPS-NP10 (http://chemgps.bmc.uu.se) to get more insights into their mechanism of action. A database of 228 cancer chemotherapeutics reported in De Ford et al.11 was used for this task. Cluster analysis was performed with CheS-Mapper49 (http:// ches-mapper.org/) to visualize the different mechanisms of action in the chemical space. The compounds were submitted in SMILES format; the eight PCs were calculated and then compared against the database. Data were analyzed as previously described.11 The 3D graphs were created with the Erl-Wood Cheminformatics 2D/3D scatterplot node of Knime. Tanimoto Coefficient Similarity Search with Knime. A data pipeline was created in Knime (Zurich, Switzerland) to calculate the molecular fingerprints using the topological path-based Indigo fingerprint node, as shown in Figure S1. The Tanimoto coefficient of the duocarmycin analogues was then calculated and compared to the database of chemotherapeutic compounds from De Ford et al.11 and to all the drugs deposited in the Drugbank19 (http://www. 24



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propanal were added to the samples in order to initiate the enzymatic reaction. The product formation of NADH was monitored by measuring the absorption increase at λ = 340 nm at 37 °C in 96-well plates with an Infinite 200 PRO NanoQuant microplate reader. All measurements were carried out in technical triplicates and biological duplicates. To determine the IC50, the log 10 of the inhibitor concentration was plotted against the remaining enzyme activity. Cell Lines and Cultivation. CCRF-CEM and CEM-ADR5000 cells were obtained as a gift from Prof. T. Efferth, Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany; Jurkat cells from ATCC (clone E6-1, ATCC TIB-152); and human lung adenocarcinoma type-II alveolar epithelial cells (A549) from the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany). MIA-PaCa-2 cells were kindly provided by Dr. Ralph Graeser (Tumour Biology Center, Freiburg, Germany). PBMC were isolated from human buffy coats obtained from the Freiburg University Clinic, Freiburg, Germany (ethical permission number from the ethics commission, AlbertLudwigs University, Freiburg: 356/13; 2013). All cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C under 5% CO2. Cell Viability Assay (MTT). Cell viability of bifunctional duocarmycin analogues 2 and 10 was tested against a small panel of cancer cell lines comprising T-cell acute lymphoblastic leukemia (CCRF-CEM, CEM-ADR-5000, and Jurkat cells), non-small-cell lung cancer (A549), and pancreatic carcinoma (MIA-PaCa-2), as well as peripheral blood mononuclear cells from healthy human donors using the MTT assay as described in Calderon et al.52 Briefly, cells were seeded in 96-well plates and incubated for 24 h with various concentrations of 2 or 10. A 25 μL amount of an MTT solution (5 mg mL−1) was added to each well, and cells were incubated for 2 h more. The IC50 values were obtained by nonlinear regression using the GraphPad Prism 5 program (Intuitive Software for Science, San Diego, CA, USA). The data are expressed as means ± SD of three independent experiments. Cell Cycle Analysis by FACS. A total of 1 ×106 cells were seeded in 12-well plates and incubated overnight. Cells were exposed to increasing concentrations of bifunctional duocarmycin analogues or 100 nM axitinib for 24 h at 37 °C and 5% CO2. Afterward, cells were fixed with cold ethanol (70%) and stained with propidium iodide (PI). The cells were incubated for 30 min with the above-mentioned conditions, and the DNA content was measured by a FACScalibur (BD Biosciences, San José, CA, USA). Ten thousand gated events were analyzed for each sample. Synthesis and Structure Determination of Bifunctional Duocarmycin Analogue 10. Detailed information can be found in the Supporting Information Statistical Analysis. Data are expressed as means ± SD. Three independent experiments were carried out with all the abovementioned methods unless otherwise stated. The Manhattan and Euclidean distance calculation was carried out with the corresponding nodes on Knime. Arbitrary cutoff values were selected to further analyze the data.

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ORCID

Lutz F. Tietze: 0000-0003-3847-0756 Irmgard Merfort: 0000-0003-4716-5016 Author Contributions

§ C. De Ford and K. Penchalaiah contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. T. Efferth, Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany, for providing CCRF-CEM and CEMADR5000 cells as a gift, and to Dr. R. Graeser (formerly Tumour Biology Center, Freiburg, Germany) for the MIAPaCa-2 cells. C.D.F. is grateful for a DAAD doctoral fellowship. This research was partly supported by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00233.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

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Bifunctional Duocarmycin Analogues as Inhibitors of Protein

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