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Minimizing higher-order aggregation maximizes iron mobilization by small molecules

Nature Chemical Biology (2024)Cite this article

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Abstract

The natural product hinokitiol mobilizes iron across lipid bilayers at low concentrations and restores hemoglobinization in iron transporter protein-deficient systems. But hinokitiol fails to similarly mobilize iron at higher concentrations, limiting its uses in chemical biology and medicine. Here we show that at higher concentrations, hinokitiol3:Fe(III) complexes form large, higher-order aggregates, leading to loss of transmembrane iron mobilization. Guided by this understanding and systematic structure–function studies enabled by modular synthesis, we identified FeM-1269, which minimally aggregates and dose-dependently mobilizes iron across lipid bilayers even at very high concentrations. In contrast to hinokitiol, FeM-1269 is also well-tolerated in animals at high doses for extended periods of time. In a mouse model of anemia of inflammation, FeM-1269 increases serum iron, transferrin saturation, hemoglobin and hematocrit. This rationally developed iron-mobilizing small molecule has enhanced potential as a molecular prosthetic for understanding and potentially treating iron transporter deficiencies.

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Fig. 1: Constitutional isomers with divergent mobilization and higher-order aggregation profiles.
Fig. 2: Structure–activity study of alkyl tropolones to identify favorable liposome and Caco-2 iron mobilization profiles.
Fig. 3: Further structure–activity study to identify and validate FeM-1269 as an optimized iron mobilizer.
Fig. 4: FeM-1269 is better tolerated than hinokitiol or GT in rats.
Fig. 5: High exposures of FeM-1269 do not disturb trace metal homeostasis.
Fig. 6: FeM-1269 promotes restored iron mobilization in AoI.

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Data availability

All data are available in the main text, extended data or supplementary materials. Source data are available through the publisher for all data in the main text and extended data figures. Any other data are available from the author(s) upon reasonable request. Crystallographic information has been deposited at the Cambridge Crystallographic Data Center 2088858 for the GT3:Fe(III) structure disclosed here. Source data are provided with this paper.

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Acknowledgements

We thank R.S. Chorghade for helpful discussions around liposome experiments, S. Robinson (Beckman Imaging Technology Group) for assistance with TEM imaging, T. Woods (George R. Clark X-Ray Facility and 3M Materials Laboratory) for X-ray structure determination, J. Katzenellenbogen for the use of a liquid scintillation counter, A.S. Grillo, A.G. Cioffi and D.J. Blair for their work on the early synthesis of tropolone derivatives and C. Murphy for the use of a Zetasizer Nano for DLS studies. TEM images were taken at the Beckman Institute for Advanced Science & Technology Imaging Technology Group (ITG) Microscopy Suite on a Thermo Fisher Scientific FEI Tecnai G2 F20 S-TWIN 200 kV STEM. The Bruker 500 MHz nuclear magnetic resonance (NMR) spectrometer was obtained with the financial support of the Roy J. Carver Charitable Trust, Muscatine, Iowa, USA. Metal analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center and was funded by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust and supported by the Office Of The Director, National Institutes of Health of the National Institutes of Health under award S10OD020118. Portions of this work were supported by the National Institutes of Health (T32GM070421 (to A.D.B.), R01HL140526 (to Y.A.S. and M.D.B.) and R35GM118185 (to M.D.B.)).

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Authors and Affiliations

  1. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Andrew D. Blake, Anna M. SantaMaria, Stella Ekaputri & Martin D. Burke

  2. Ambys Medicines, South San Francisco, CA, USA

    Jianhua Chao, Nicholas M. Snead, Douglas M. Matje, Tao Geng, Angela Octaviani & Stanley J. Hollenbach

  3. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Kelsie J. Green, Samantha T. Brown, Christopher K. Rakowski, Peng-Jui Chen & Martin D. Burke

  4. Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, MI, USA

    Eun-Kyung Choi, Luisa Aring & Young-Ah Seo

  5. Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA

    Keith Bailey

  6. Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Timothy M. Fan

  7. Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Timothy M. Fan & Martin D. Burke

  8. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Martin D. Burke

  9. Molecule Maker Lab Institute, Arnold and Mabel Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Martin D. Burke

  10. Carle Illinois College of Medicine, Champaign, IL, USA

    Martin D. Burke

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Contributions

A.D.B., A.M.S.M. and M.D.B. designed the study. A.D.B. performed liposome efflux, Caco-2, DLS, TEM, X-ray crystallographic analysis and statistical analyses. A.M.S.M. and K.J.G. performed Caco-2 studies. A.D.B. and P.-J.C. synthesized and evaluated the initial set of alkyl tropolonoids. J.C. and D.M.M. conducted the medicinal chemistry campaign that identified FeM-1269. N.M.S., T.G. and A.O. designed, coordinated and performed animal studies and performed statistical analyses. C.K.R. and S.T.B. performed mechanistic DLS studies. C.K.R. and S.E. performed studies comparing iron chelators and iron mobilizers. E.-K.C., L.A. and Y.A.S. performed studies in the flatiron mouse anemia model. T.F. performed the mouse study assessing stress erythropoiesis, assisted by S.E. and K.J. G. S.J.H., Y.A.S. and M.D.B. oversaw the work described in this study. A.D.B. and M.D.B. wrote the paper, which was reviewed by all authors.

Corresponding authors

Correspondence to Young-Ah Seo or Martin D. Burke.

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Competing interests

M.D.B. is an inventor on the US patent application pub. 20180263926, ‘Restoring Physiology in Iron-Deficient Organisms Using Small Molecules’, which is directed to the use of small-molecule iron transporters to treat deficiencies of iron-transport proteins. A.D.B., A.M.S.M. and M.D.B. are inventors on the US patent application pub. 20210163393, ‘Hinokitiol analogs, methods of preparing and pharmaceutical compositions thereof’, which is directed to analogs of hinokitiol, preparation and formulation. These patent applications, their foreign counterparts and others have been licensed by Cajal Neuroscience, for which M.D.B. is a scientific advisor and shareholder. Ambys Medicines has filed patent applications directed to tropolone derivatives and tautomers thereof for iron regulation in animals. J.C., N.M.S., D.M.M., T.G., A.O. and S.J.H. were full-time employees of Ambys Medicines when these data were generated. These patents are currently owned by Cajal Neuroscience. The other authors declare no competing interests.

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Nature Chemical Biology thanks Christopher Chang and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In vitro iron mobilization and aggregation by tropolonoids.

a, Time course of iron efflux from liposomes with different concentrations of hinokitiol, GT or FeM-1269 (n = 3; representative experiment shown). b, Rates of iron efflux from liposomes at each concentration of compounds, calculated from data in a–c. c, DLS derived count rate measured in different concentrations of MES/TRIS buffer, pH 7.0 (n = 3; representative experiment shown). d, DLS derived count rate measured at different pH of 50 mM MES/TRIS buffer (n = 3; representative experiment shown). e, DLS derived count rate measured with different buffering agents at 50 mM of buffer (n = 3; representative experiment shown). f, DLS derived count rate of hinokitiol2:Cu(II), GT2:Cu(II) and FeM-12692:Cu(II) (n = 3; representative experiment shown). g, Additional representative images from 90 μM of hino3:Fe(III), GT3:Fe(III) or FeM-12693:Fe(III) in buffer (n = 50, 45, 51 images for hino, GT and FeM-1269, respectively; images were taken from three independent sample preparations; scale bar is 500 nm). h, Iron efflux by tropolone derivatives from shDMT1 Caco-2 epithelia at 1 μM (n = 3, representative experiment shown). i, Iron efflux by tropolone derivatives from shDMT1 Caco-2 epithelia at 5 μM (n = 3, representative experiment shown).

Source data

Extended Data Fig. 2 Physicochemical parameters of tropolone iron mobilizer library.

a, Physicochemical parameters of tropolones relative to performance in liposomes, defined by Elip. b, Physicochemical parameters of tropolones relative to iron mobilized in shDMT1-Caco-2 differentiated gut epithelia after 4 hours at 3 μM, tracked by 55FeCl3 (n = 3; experiments were performed at least twice). c, Physicochemical parameters of tropolones relative to iron mobilized in shDMT1-Caco-2 differentiated gut epithelia after 4 hours at 10 μM, tracked by 55FeCl3 (n = 3; experiments were performed at least twice). All parameters were computed in SwissADME.

Source data

Extended Data Fig. 3 Physicochemical parameters and chemical structures of highly effective iron mobilizers.

a–f, Physicochemical parameters of top tropolones in each assay for iron mobilization (n = 20 tropolones). Extracted from the datasets displayed in Extended Data Fig. 2. g, Chemical structures of the top 20 tropolone iron mobilizers in shDMT1-Caco-2 epithelia at 10 μΜ are largely substituted with short alkyl- or alkoxy-side chain substituents. Of note, (+)-6 and (−)-6 are isolated during the preparation of (±)-6 and were independently evaluated. See the Supplementary Note for details on chiral separation. Error bars indicate SEM.

Source data

Extended Data Fig. 4 FeM-1269 has a favorable drug-like profile.

a, Efflux of Fe(III) after 30 minutes, tracked by incorporation into ferrozine (n = 3; representative experiment shown). b, Iron efflux from shDMT1 Caco-2 at several concentrations (n = 3; representative experiment shown). c, Fe(III) efflux from FPN1-KO RAW264.7 macrophages after 2 hours, tracked by 55FeCl3 (n = 3; representative experiment shown). Error bars indicate SEM. d, Dashboard of in vitro ADME studies on FeM-1269. e, Stoichiometric competition experiment with 1 mM of each divalent metal and 1 mM of the indicated tropolone. Tropolone-bound metal was measured by ICP-MS after equilibration and extraction with ethyl acetate (n = 3 independent samples). f, Serum plasma content in mice of tropolonoids after single-dose of 10 mg/kg in water for injection (n = 3 animals per group). g, Calculated pharmacokinetic parameters of data in c. h, Maximum tolerated dose finding and pharmacokinetic study in mice of orally administered FeM-1269 (n = 2 animals per group) – MTD = 1000 mg/kg. i, Correlation between exposure and administered dose, using data in Fig. 4d and the study in f. j, Calculated pharmacokinetic calculations for MTD study in e (n = 2 per group). k, Results from micronucleus testing at MTD of FeM-1269 of 1000 mg/kg/day – (n = 5, 5, 4 animals for vehicle treated, 24 hours post-FeM-1269 and 48 hours post-FeM-1269, respectively). l, Pharmacokinetic study of FeM-1269 in rats (n = 3 animals per group). m, Pharmacokinetic study of FeM-1269 in beagle dogs (n = 3 animals per group). Detailed pharmacokinetic calculations are available in Supplementary Table 3 for the studies in l,m. Error bars indicate SEM.

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Extended Data Fig. 5 Additional outcomes from two-week toxicology studies with hinokitiol and GT.

a, Animal weights from two-week daily administration of hinokitiol at 10, 30 and 100 mg/kg/day (n = 6 animals per group). b, Pharmacokinetic study to establish bioavailability and approximate exposure to hinokitiol (n = 3 animals per group). c, Pharmacokinetics of hinokitiol administered on day 1 to rats (n = 3 animals per group). d, Pharmacokinetics of hinokitiol administered on day 15 to rats (n = 3 animals per group). e, Pharmacokinetic calculations from studies in c,d. f, Representative H&E staining of rat myocardium from an animal treated with 30 mg/kg of hinokitiol. g, Representative H&E staining of rat myocardium from an animal treated with 100 mg/kg of hinokitiol. h, Representative H&E staining of rat myocardium from an animal treated with 100 mg/kg of hinokitiol. i, Rat weights from two-week daily administration of GT at 15, 45 and 105 mg/kg/day (n = 6 animals per group). j, Pharmacokinetic study to establish bioavailability and approximate exposure to GT (n = 3 animals per group). k, Pharmacokinetic study of serum concentrations of GT on day 1 of the study (n = 3 animals per group). l, Pharmacokinetic study of serum concentrations of GT on day 15 of the study (n = 3 animals per group). m, Pharmacokinetic calculations from studies with GT in c,d. n, Representative H&E staining of rat myocardium from an animal treated with 45 mg/kg of GT. o, Representative H&E staining of rat myocardium from an animal treated with 105 mg/kg of GT. p, Representative DAB-Perl’s staining of rat myocardium from an animal treated with 105 mg/kg of GT. Arrows indicate features of interest. For panels f–h, n–p, the scale bars indicate 200 μm, and a minimum of three animals were processed and imaged for each treatment group. Pharmacokinetic calculations were performed in WinNonlin. Error bars indicate SEM.

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Extended Data Fig. 6 Additional data from two-week rat toxicology study with FeM-1269.

a, Animal weights from two-week daily administration of FeM-1269 at 30, 100 and 300 mg/kg/day in male rats (n = 3 animals per group). b, Animal weights from two-week daily administration of FeM-1269 at 30, 100 and 300 mg/kg/day in female rats (n = 3 animals per group). c, Pharmacokinetic study of serum concentrations of FeM-1269 on day 1 of the study in male rats (n = 2 animals per group). d, Pharmacokinetic study of serum concentrations of FeM-1269 on day 15 of the study in males (n = 2 animals per group). e, Pharmacokinetic study of serum concentrations of FeM-1269 on day 1 of the study in females (n = 2 animals per group). f, Pharmacokinetic study of serum concentrations of FeM-1269 on day 15 of the study in females (n = 2 animals per group). g, Calculated pharmacokinetic parameters from studies in c–f. Error bars indicate SEM.

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Extended Data Fig. 7 Additional data from three-month mouse toxicology studies with FeM-1269 and stress erythropoiesis follow-up.

a, Male weights with daily administration of FeM-1269 (n = 6 animals per group). b, Female weights with daily administration of FeM-1269 (n = 6 animals per group). c,d, Histopathological analysis of tissues following FeM-1269 administration for 90 days in male and female mice (n = 6 animals per group). Only the tissues in which findings were observed were included in these graphs. Observation of the following tissues yielded no histopathological findings: brain, skeletal muscle, small intestine, spleen or thymus. Statistics in c,d were performed using two-way ANOVA with Dunnett’s multiple comparisons test. e, Female mouse weights following treatment with vehicle (0.5% HPMC) or 150 mg/kg/day of FeM-1269 (n = 12 animals per group). f, Spleen iron as assessed by ICP-MS (n = 12 per group, p = 0.0437). g, Histopathology analysis of mouse spleens using Prussian blue staining (n = 12 per group, p = 0.1114). h, Counts of bone marrow burst-forming unit, erythrocytes (BFU) (n = 12 per group, p = 0.4848). i, Counts of bone marrow colony-forming unit, erythrocytes (CFU) (n = 12 per group, p = 0.6243). j, Counts of spleen-derived BFU (n = 12 per group, p = 0.9999). k, Counts of bone marrow burst-forming unit, erythrocytes (CFU) (n = 12 per group, p = 0.0934). Statistics in f–k were performed using two-sided unpaired t-tests with Welch’s correction. For p-values for c,d, please see the source data file. Error bars represent SEM.

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Extended Data Fig. 8 Studies of FeM-1269 administration in flatiron mice.

In a–d, animals were administered vehicle or increasing doses of FeM-1269 via oral gavage and sacrificed 3 hours later for data collection (n = 6 animals for all groups). a, Duodenum non-heme iron levels. b, Spleen non-heme iron levels. c, Liver non-heme iron levels. d, Transferrin saturation. In e–k, animals were given 10 mg/kg of either hinokitiol, FeM-1269 or deferiprone daily for seven days, then sacrificed three hours after the final dose for data collection (n = 6 animals for WT + vehicle, FeM-1269 10 mg/kg; n = 5 for other groups). e, Duodenum non-heme iron levels (n = 6 animals for WT + vehicle, FeM-1269 10 mg/kg; n = 5 for other groups). f, Spleen non-heme iron levels. g, Liver non-heme iron levels. h, Transferrin saturation levels. i, Total serum iron levels. j, Hemoglobin. k, Hematocrit. Statistics were performed using one-way ANOVA with Tukey’s multiple comparisons test between each group and the vehicle treated group. For p-values, please see the source data file. Error bars indicate SEM. Where displayed, *p < 0.05 relative to vehicle controls.

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Extended Data Fig. 9 Additional data from anemia of inflammation study in Fig. 6.

In a–f, mice were treated as described in the supporting information section ‘Anemia of Inflammation Dose-Escalation Study’ (n = 10 animals for Sham and TO + bleed groups; n = 12 for all other groups). a, Weight of mice employed in the study. b, Calculated daily exposure to FeM-1269 based on pharmacokinetic data in Extended Data Fig. 4i. c, Transferrin saturation following daily administrations of hinokitiol (predicted area under the curve (AUC) 9560 ng/mL * hr). d, Total serum iron following daily administration of hinokitiol. e, Hemoglobin following daily administration of hinokitiol. f, Hematocrit following daily administration of hinokitiol. Sham and vehicle data in c–f are replicated from Fig. 6. Statistics were analyzed in Prism using one-way ANOVA with Dunnett’s or Tukey’s multiple comparisons tests as appropriate. For p-values, please see the source data file. Error bars indicate SEM. Where displayed, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 relative to vehicle controls or as otherwise specified.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–25, Supplementary Tables 1–51 and Supplementary Note.

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Blake, A.D., Chao, J., SantaMaria, A.M. et al. Minimizing higher-order aggregation maximizes iron mobilization by small molecules. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01596-3

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