Rational drug design, synthesis, and biological evaluation of novel chiral tetrahydronaphthalene-fused spirooxindole as MDM2-CDK4 dual inhibitor against glioblastoma

Cells treated with ent-4g showed up-regulation of proteins involved in P53 and cell cycle pathways.

“Cells treated with ent-4g showed up-regulation of proteins involved in P53 and cell cycle pathways.”

ent-4g showed good anti-tumor efficacy against glioblastoma xenografts in mice.

“The compound showed good anti-tumor efficacy against glioblastoma xenografts in mice.”

• Also stated in: § Ent-4g inhibits U251 glioblastoma cell proliferation by altering cell cycle progression and P53 signalling · plain

  “All treatments potently inhibited tumor growth, with ent-4g showing significantly greater effects than the reference drugs.”

Selectivity differed by over 40-fold between the least and most potent stereoisomers in TR-FRET and KINOMEscan in vitro assays.

“selectivity differed by over 40-fold between the least and most potent stereoisomers in time-resolved FRET and KINOMEscan® in vitro assays.”

Analysis of genomic alterations in glioblastoma according to TCGA identified the q13-15 region of chromosome 12 as one of the regions most often rearranged in the disease.

“Our analysis of genomic alterations in glioblastoma according to data in the Cancer Genome Atlas (TCGA) identified the q13–15 region of chromosome 12 as one of the regions that most often rearranged in the disease (Fig. 1A and B)5, 6, 7.”

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Both MDM2 and CDK4 genes are significantly overexpressed at the mRNA and protein levels in patients with glioblastoma, regardless of P53 mutation status.

“We also verified both genes to be significantly overexpressed at the mRNA and protein levels in patients with glioblastoma, regardless of P53 mutation status (Fig. 1C–E).”

Binding conformation differed substantially between 4g/4g′ and ent-4g/ent-4g′.

“Binding conformation differed substantially between 4g/4g′ and ent-4g/ent-4g′; during the dynamic's simulation, 4g moved to another ANS binding site, 4g′ moved to the ATP binding site and ent-4g′ moved to the hydrophobic pocket.”

ent-4g inhibited the growth of glioblastoma xenografts expressing mutant P53 better than the MDM2 inhibitor nutlin-3a alone or together with palbociclib.

“The novel compound inhibited the growth of glioblastoma xenografts expressing mutant P53 better than the MDM2 inhibitor nutlin-3a alone or together with palbociclib.”

Isomers ent-4d and ent-4g inhibited growth of U251 cells better than palbociclib.

“The isomers ent-4d and ent-4g inhibited growth of U87MG cells to a greater extent than nutlin-3a or palbociclib, and they inhibited growth of U251 cells better than palbociclib.”

CDK4 is overexpressed in over half of patients with glioblastoma associated with mutations in P53.

“CDK4 is the most often overexpressed CDKs in the disease, and it is overexpressed in over half of patients with glioblastoma associated with mutations in P53 (Fig. 1D).”

ent-4g treatment triggered an increase in MDM2, P53 and P21 levels.

“The compound treatment triggered an increase in MDM2, P53 and P21 levels (Fig. 4E and F).”

Enrichment analysis using integrated GO, KEGG and Biocarta revealed significant alteration in the cell cycle and P53 signalling pathways.

“Enrichment analysis using integrated GO103, KEGG104 and Biocarta105 revealed significant alteration in the cell cycle and P53 signalling pathways, as shown in the KEGG pathway enrichment results (Fig. 4B).”

• cites GO103Not checked yet

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• cites KEGG104Not checked yet

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• cites Biocarta105Not checked yet

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ent-4g caused negligible or minimal inhibition to most kinases other than CDK4-cyclinD1, CDK4 and CDK2.

“The compound caused negligible or minimal inhibition to most kinases other than CDK4-cyclinD1, CDK4 and CDK2.”

At high concentrations, all compounds showed good inhibition of two cell lines expressing mutated P53 (T98G and U251) and one cell line expressing wild-type P53 (U87MG).

“At high concentrations, all compounds showed good inhibition of two cell lines expressing mutated P53 (T98G and U251) and one cell line expressing wild-type P53 (U87MG, Fig. 3B, C).”

Analysis of CDK expression and mutations in glioblastoma samples in TCGA indicates that CDK4 is the most often overexpressed CDK in the disease.

“Analysis of CDK expression and mutations in glioblastoma samples in TCGA database indicates that CDK4 is the most often overexpressed CDKs in the disease, and it is overexpressed in over half of patients with glioblastoma associated with mutations in P53 (Fig. 1D).”

The highest binding free energy changes were the mutation of Leu54 to alanine in both ent-4g and SAR405838 complexed to MDM2.

“The highest binding free energy changes were the mutation of Leu54 to alanine in both ent-4g and SAR405838 complexed to MDM2, the Thr16 in ent-4g complex and Lys94 in SAR405838 complex were also stronger than the other residues (>4.0 kcal/mol).”

• AssumptionThe authors state this as an assumption: Local changes of the protein do not influence the whole conformation of the complex significantly.

ent-4g showed no appreciable inhibition of other CDKs or kinases.

“it showed nanomolar IC50 against CDK4, micromolar IC50 against CDK2, and no appreciable inhibition of other CDKs or kinases.”

Among the more active compounds 4a-4j, derivatives 4d and 4g with a halogen at the 5-position of the oxindole showed the greatest MDM2 inhibition and cytotoxicity.

“Among the more active compounds 4a–4j, derivatives 4d and 4g with a halogen at the 5-position of the oxindole showed the greatest MDM2 inhibition and cytotoxicity.”

Compounds 4a-4p worked better than compounds 4a′-4p′ at inhibiting the activity of MDM2 and CDK4 as well as the proliferation of glioblastoma cell lines.

“Compounds 4a–4p worked better than compounds 4a′–4p′ at inhibiting the activity of MDM2 and CDK4 as well as the proliferation of glioblastoma cell lines.”

Preliminary docking studies and integrative molecular simulations suggested that an inhibitor bearing a planar THN would bind well to CDK4 and MDM2.

“Preliminary docking studies and integrative molecular simulations suggested that an inhibitor bearing a planar THN would bind well to CDK4 and MDM2.”

Isomers ent-4d and ent-4g inhibited growth of U87MG cells to a greater extent than nutlin-3a or palbociclib.

“The isomers ent-4d and ent-4g inhibited growth of U87MG cells to a greater extent than nutlin-3a or palbociclib, and they inhibited growth of U251 cells better than palbociclib.”

Cell proliferation inhibitory potencies of compounds 4d-4h in U87MG cells were better than that of T98G and U251 cells.

“It was notable that the cell proliferation inhibitory potencies of compounds 4d–4h in U87MG cells were better than that of T98G and U251 cells, which suggested that only the activation of”

4d and 4g inhibited MDM2 and CDK4 less than nutlin-3a and palbociclib.

“Although 4d and 4g inhibited MDM2 and CDK4 less than nutlin-3a and palbociclib, all compounds showed similar cytotoxicity against the tested glioblastoma cell lines based on the MTT assay.”

ent-4g increased the proportion of glioblastoma cells showing hyper-condensed, apoptotic nuclei based on Hoechst 33,342 staining.

“In addition, ent-4g increased the proportion of glioblastoma cells showing hyper-condensed, apoptotic nuclei based on Hoechst 33,342 staining (Beyotime, Shanghai, China, Supporting Information Fig. S5).”

Mutation of key binding residues resulted in significant increase of binding free energies, suggesting disrupted inhibitor-residue interactions.

“As was expected, the mutation of key binding residues resulted significant increase of binding free energies, which suggested the disrupted inhibitor–residue interactions.”

• AssumptionThe authors state this as an assumption: Local changes of the protein do not influence the whole conformation of the complex significantly.

All compounds showed similar cytotoxicity against the tested glioblastoma cell lines based on the MTT assay.

“Although 4d and 4g inhibited MDM2 and CDK4 less than nutlin-3a and palbociclib, all compounds showed similar cytotoxicity against the tested glioblastoma cell lines based on the MTT assay.”

In the case of CDK2, 4% of control protein remained after competitive binding of 100 nmol/L ent-4g (0.8% to CDK4-cyclinD1 and 2.2% to CDK4).

“In the case of CDK2, 4% of control protein remained after competitive binding of 100 nmol/L ent-4g (0.8% to CDK4-cyclinD1 and 2.2% to CDK4), which is probably because CDK2 possesses 66% of sequence identities to CDK4.”

The HR-MS of compound 4n' showed a found m/z of 550.0187 for [M+Na]+, matching the calculated 550.0185 for C24H15N3O7Cl2Na.

“HR-MS (ESI): m/z Calcd. for C24H15N3O7Cl2Na [M+Na]+: 550.0185, Found 550.0187.”

Compound 4d′ was obtained with 95% enantiomeric excess.

“White solid, 76% yield (28.1 mg, 0.06 mmol), ee 95%, [α]D25 = +48.72 (c 0.10 in CH2Cl2), ent-4d′: –37.99, m.p.>220 °C.”

H&E staining of tissue sections from main organs after treatment indicated no severe toxic effects.

“Despite these anti-tumor effects of ent-4g, hematoxylin and eosin staining of tissue sections from main organs after treatment indicated no severe toxic effects (Supporting Information Fig. S6).”

Like palbociclib, ent-4g inhibited autophosphorylation of CDK4 and phosphorylation of retinoblastoma (RB) in U251 cells.

“Like palbociclib, ent-4g inhibited autophosphorylation of CDK4 and phosphorylation of retinoblastoma (RB) in U251 cells (Fig. 4E).”

Compound 4g′ was obtained with 95% enantiomeric excess.

“White solid, 78% yield (29.5 mg, 0.06 mmol), ee 95%, [α]D25 = +38.53 (c 0.10 in CH2Cl2), ent-4g′: –31.00, m.p. >220 °C.”

ent-4g stimulated BAX to a greater extent than nutlin-3a did.

“In fact, the compound stimulated BAX to a greater extent than nutlin-3a did, and it activated more cleavage of caspase-3 than palbociclib did.”

Compound 4g was obtained with 95% enantiomeric excess.

“White solid, 88% yield (32.8 mg, 0.06 mmol), ee 95%, [α]D25 = –99.97 (c 0.10 in CH2Cl2), ent-4g: +109.98, m.p.>220 °C.”

ent-4g activated more cleavage of caspase-3 than palbociclib did.

“In fact, the compound stimulated BAX to a greater extent than nutlin-3a did, and it activated more cleavage of caspase-3 than palbociclib did.”

The absolute oral bioavailability of ent-4g was around 30%.

“and the absolute oral bioavailability of ent-4g was around 30%.”

ent-4g showed strong ability to inhibit both MDM2 and CDK4 in glioblastoma cells expressing wild-type or mutant P53.

“The compound ent-4g showed strong ability to inhibit both MDM2 and CDK4 in glioblastoma cells expressing wild-type or mutant P53.”

The results reveal enhanced tumor exposure of ent-4g compared to plasma.

“The results in Supporting Information Fig. S7 reveal the enhanced tumor exposure of ent-4g compared to plasma.”

ent-4g displayed good stability in human liver microsomes assay, with over 90% of ent-4g remaining after 10 min incubation of 1 mg/mL proteins at 37 °C.

“Moreover, compound ent-4g displayed good stability in human liver microsomes assay, with over 90% of ent-4g remained after 10 min incubation of 1 mg/mL proteins at 37 °C, and its half-life period in human liver microsomes assay was 46.5 min.”

ent-4g strongly inhibited the growth of glioblastoma xenografts in mice.

“The compound also strongly inhibited the growth of glioblastoma xenografts in mice.”

The HR-MS of compound 4o' showed a found m/z of 560.0070 for [M+Na]+, matching the calculated 560.0069 for C24H16N3O7BrNa.

“HR-MS (ESI): m/z Calcd. for C24H16N3O7BrNa [M+Na]+: 560.0069, Found 560.0070.”

The HR-MS of compound 4p' showed a found m/z of 512.1069 for [M+Na]+, matching the calculated 512.1070 for C25H19N3O8Na.

“HR-MS (ESI): m/z Calcd. for C25H19N3O8Na [M+Na]+: 512.1070, Found 512.1069.”

The half-life period of ent-4g in human liver microsomes assay was 46.5 min.

“and its half-life period in human liver microsomes assay was 46.5 min.”

Compound 4d was obtained with 99% enantiomeric excess.

“White solid, 87% yield (31.6 mg, 0.07 mmol), ee 99%, [α]D25 = –75.99 (c 0.10 in CH2Cl2), ent-4d: +67.89, m.p.>220 °C.”

Glioblastoma involves deregulation of P53 and cell cycle signalling pathways.

“The disease involves deregulation of P53 and cell cycle signalling pathways1, 2, 3, 4.”

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Rational design, asymmetric synthesis and biological evaluation of novel THN-fused spirooxindoles could generate promising MDM2-CDK4 dual inhibitors in glioblastoma therapy.

“These results suggested that rational design, asymmetric synthesis and biological evaluation of novel tetrahydronaphthalene fused spirooxindoles could generate promising MDM2-CDK4 dual inhibitors in glioblastoma therapy.”

• AssumptionRests on an assumption: Investigation of all-carbocycle modifications at the C3 position as potent MDM2 inhibitors is underdeveloped.

Simultaneous inhibition of MDM2 and CDK4 may be an effective treatment against glioblastoma.

“Simultaneous inhibition of MDM2 and CDK4 may be an effective treatment against glioblastoma.”

• Also stated in: § Introduction · hedged

  “These results suggest that simultaneous inhibition of both MDM2 and CDK4 may be effective against glioblastoma36, 37, 38, 39.”

Appropriate stereochemistry in THN-fused spirooxindole compounds is key to their inhibitory activity.

“Appropriate stereochemistry in THN-fused spirooxindole compounds is key to their inhibitory activity: selectivity differed by over 40-fold between the least and most potent stereoisomers in time-resolved FRET and KINOMEscan® in vitro assays.”

At the P53-binding site in MDM2, the THN-fused C3-spirooxindole could form hydrogen bonds and hydrophobic interactions mimicking Phe19, Trp23 and Leu25 of P53.

“At the P53-binding site in MDM2, the THN-fused C3-spirooxindole could form hydrogen bonds and hydrophobic interactions mimicking Phe19, Trp23 and Leu25 of P53.”

Only the activation of wild-type P53 should suppress glioblastoma cell proliferation.

“which suggested that only the activation of wild-type P53 should suppress the glioblastoma cell proliferation.”

THN-fused C3-spirooxindoles should possess good druglikeness because THN and spirooxindole derivatives are privileged drug-like architectures.

“We started with THN54, 55, 56, 57, 58, 59, 60, 61 and spirooxindole derivatives62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 because they are privileged drug-like architectures, so the resulting THN-fused C3-spirooxindoles should possess good druglikeness (Fig. 2C).”

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CDK4 is substantially overexpressed in glioblastoma, breast and ovarian cancers.

“CDK4, one of the main controllers of cell cycle entry, is substantially overexpressed in glioblastoma, breast and ovarian cancers, making it an attractive therapeutic target27, 28, 29.”

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In spirocyclic oxindole-based MDM2 inhibitors, the oxindole fragment occupies the Trp23-containing cleft of P53.

“In spirocyclic oxindole-based MDM2 inhibitors, the oxindole fragment occupies the Trp23-containing cleft of P53, and appropriate stereochemistry is critical for good binding affinity73,74.”

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The stable hydrogen bond between ent-4g and MDM2 residue Thr16 has never been reported before.

“and that the compound forms a stable hydrogen bond with MDM2 residue Thr16 (Fig. 3G), which has never been reported before.”

Computational alanine scanning results confirmed the binding modes of ent-4g suggested by molecular docking and MD simulation.

“The computational alanine scanning results also confirmed that binding modes of ent-4g suggested by molecular docking and MD simulation.”

• AssumptionThe authors state this as an assumption: Local changes of the protein do not influence the whole conformation of the complex significantly.

ent-4g can be regarded as a specific MDM2/CDK4 inhibitor.

“These results suggest that ent-4g can be regarded as a specific MDM2/CDK4 inhibitor.”

Compound ent-4g displayed good CDK selectivity.

“In particular, compound ent-4g displayed good CDK selectivity: it showed nanomolar IC50 against CDK4, micromolar IC50 against CDK2, and no appreciable inhibition of other CDKs or kinases.”

The regulation mechanisms between MDM2 and CDK4 may be more complicated than previously thought.

“These results indicated that the regulation mechanisms between MDM2 and CDK4 may be more complicated than previously thought.”

Simple combination therapy of MDM2 and CDK4 inhibitors in preclinical experiments demonstrated paradoxical results in sarcoma.

“Although the simple combination therapy of MDM2 and CDK4 inhibitors in preclinical experiments were reported recently, the two independent reports demonstrated the paradoxical results in sarcoma51,52.”

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In the CDK4 allosteric site, the scaffold could interact with surrounding hydrophobic residues and residues in the DFG-loop, avoiding interactions with the highly conserved ATP-binding site that might reduce selectivity for CDK4.

“In the CDK4 allosteric site, the scaffold could interact with surrounding hydrophobic residues and residues in the DFG-loop, avoiding interactions with the highly conserved ATP-binding site that might reduce selectivity for CDK487.”

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Co-amplification of MDM2 and CDK4 has been reported in several types of cancers including sarcoma, glioblastoma, bladder cancer and gastric cancer.

“Moreover, the co-amplification of MDM2 and CDK4 has been reported in several type of cancers including sarcoma, glioblastoma, bladder cancer, gastric cancer, etc.40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50”

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The strong cytotoxicity of ent-4g against glioblastoma cells expressing mutated P53 is consistent with its low IC50 values against MDM2 and CDK4.

“The strong cytotoxicity of ent-4g against glioblastoma cells expressing mutated P53 is consistent with its low IC50 values against MDM2 and CDK4.”

ent-4g mimics P53 residues Phe19, Trp23 and Leu25 in interacting with MDM2.

“These analyses suggest that ent-4g mimics P53 residues Phe19, Trp23 and Leu25 in interacting with MDM2, and that the compound forms a stable hydrogen bond with MDM2 residue Thr16 (Fig. 3G), which has never been reported before.”

ent-4g forms a stable hydrogen bond with MDM2 residue Thr16.

“These analyses suggest that ent-4g mimics P53 residues Phe19, Trp23 and Leu25 in interacting with MDM2, and that the compound forms a stable hydrogen bond with MDM2 residue Thr16 (Fig. 3G), which has never been reported before.”

Appropriate stereochemistry is critical for good binding affinity in spirocyclic oxindole-based MDM2 inhibitors.

“In spirocyclic oxindole-based MDM2 inhibitors, the oxindole fragment occupies the Trp23-containing cleft of P53, and appropriate stereochemistry is critical for good binding affinity73,74.”

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We have discovered THN-fused spirooxindole derivative ent-4g as a potent inhibitor through rational drug design and asymmetric synthesis.

“In summary, we have discovered THN-fused spirooxindole derivative ent-4g as a potent inhibitor through rational drug design and asymmetric synthesis of the designed compounds.”

The low plasma concentration of ent-4g (lower than 100 nmol/L after 1-h administration) suggested a potential low toxicity profile of ent-4g in vivo.

“The low plasma concentration of ent-4g (lower than 100 nmol/L after 1-h administration) suggested a potential low toxicity profile of ent-4g in vivo.”

• AssumptionRests on an assumption: A low plasma concentration of ent-4g predicts a low toxicity profile in vivo.

Molecular dynamics simulations indicate that ent-4g tightly binds to MDM2 and CDK4.

“Molecular dynamics simulations indicate that the compound ent-4g tightly binds to MDM2 and CDK4.”

Purity of compounds 4 and 4′ was determined by reverse-phase HPLC analysis to be >95% at 254 nm.

“Purity of compounds 4 and 4′ was determined by reverse-phase HPLC analysis to be >95% at 254 nm.”