Minimizing higher-order aggregation maximizes iron mobilization by small molecules

Blake, Andrew David

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https://hdl.handle.net/2142/115881 Copy

Description

Title

Minimizing higher-order aggregation maximizes iron mobilization by small molecules

Author(s)

Blake, Andrew David

Issue Date

2022-07-14

Director of Research (if dissertation) or Advisor (if thesis)

Burke, Martin D

Doctoral Committee Chair(s)

Burke, Martin D

Committee Member(s)

• Hergenrother, Paul J
• Procko, Erik
• Kalsotra, Auinash

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• molecular prosthetic
• hinokitiol
• iron mobilization

Abstract

In the classic paradigm of pharmacology, small molecules bind to and modulate protein targets to promote or inhibit activity of dysfunctional proteins to treat diseases. However, diseases caused by total lack of protein function do not have any modulable targets and are thus refractory to this approach. One such disorder is anemia of inflammation (AoI), which is the second most prevalent form of anemia and afflicts roughly one billion people. AoI is an acquired deficiency of the ferroportin (FPN1) protein, the sole known mammalian iron exporter, resulting from inflammation triggered increases in serum IL-6 and hepcidin. This results in tissue iron-retention, limiting the iron available for erythropoiesis. Known therapeutic options fail to directly address the iron transporter deficiency and tissue iron retention. To this end, the Burke group has pioneered the concept of molecular prosthetics, small molecules which autonomously replicate the function of missing proteins, as therapeutics and chemical probes for these disorders. In the case of missing iron transporter proteins, the natural product hinokitiol mobilizes iron across lipid bilayers and restores hemoglobinization in iron transporter deficient cells and animals. Site- and direction-selective transmembrane iron mobilization by an inherently non-selective small molecule was achieved by leveraging pathophysiological electrochemical gradients favoring iron mobilization across membranes lacking iron transport function. There is, however, an important limitation that diminishes hinokitiol’s utility as a molecular prosthetic. In differentiated shDMT1-Caco-2 monolayers, hinokitiol effectively mobilized iron at low-to-intermediate concentrations, but at higher concentrations transmembrane iron mobilizing activity by hinokitiol decreased. The mechanism underlying this loss of activity at higher concentrations has remained unclear. Understanding and thereby mitigating this bimodal effect is required to maximally harness the substantial potential for iron mobilizing small molecules to serve as tools for chemical biology or potential therapeutics. With the goal of understanding this concentration-dependent loss of activity, we studied two tropolone natural products, hinokitiol and -thujaplicin (GT), with similar chemical structures but different concentration-dependent transmembrane iron mobilizing effects, both in Caco-2 epithelia and iron(III)-loaded liposomes. Using multiple biophysical methods, we found that loss of hinokitiol-mediated iron mobilization at higher concentrations is paralleled by formation of large higher-order aggregates of hino3:Fe in aqueous media. GT3:Fe shows a reduced ability to form higher-order aggregates at higher concentrations and a wider range of effective concentrations. X-ray crystallographic analysis suggested that structural modification and/or changing the position of the side chain could mitigate higher-order aggregation, and thus promote concentration-dependent increases in transmembrane iron mobilizing activity. Guided by this key mechanistic insight, we embarked on a lego-like synthesis campaign to systematically analyze the impact of different tropolone substitution patterns on iron mobilization. This campaign ultimately yielded the knowledge that intermediately lipophilic tropolones robustly mobilize iron over a wide range of concentrations in both liposomes and Caco-2 epithelia. We also studied a similar suite of deferiprone analogs and found that deferiprone derivatives with intermediate lipophilicity could show similar liposome efflux characteristics. Using insights from this campaign in collaboration with a pharmaceutical start-up, a broader structure-activity study yielded AMB-1269, an optimized iron mobilizer which minimally aggregates in aqueous media and mobilizes iron even at very high concentrations. Multiple animal studies yielded promising results, including a study in a mouse model of AoI, where AMB-1269 promoted dose-dependent increases in serum iron levels and also restored hematocrit. Finally, we showed that hinokitiol, which also binds to and mobilizes copper, is competent to restore physiology in copper transport deficient systems. Thus, the mechanistic insights described herein enabled the rational development of a novel troponoloid with increased potential to serve as a molecular prosthetic to treat AoI and other anemias caused by loss of FPN1 function.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Andrew Blake

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