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Ferrous iron-dependent pharmacology and drug delivery

Improved antimalarials

A long-standing research interest of the Renslo Lab is the design and synthesis of drug-like molecules that exhibit ferrous iron-dependent pharmacology. In one aspect of this work, we are seeking to improve the in vivo efficacy and antiparasitic spectrum of existing antimalarial drugs like artemisinin and artefenomel, endoperoxides that become activated by free Fe(II) sources in the malaria parasite during blood-stage infection. Based on a conformational understanding of Fe(II)-promoted reactivity, we are currently exploring closely related but hitherto underexplored 1,2,4-trioxolane chemotypes TRX-A/C/P (shown below) that we predict should combine improved Fe(II) stability in vivo with enhanced activity against P. falciparum parasites, including emerging artemisinin-resistant strains. Already this approach has yielded novel analogs that confer single-dose cures in animal models of malaria.


Antimalarial trioxolanes: Activation of antimalarial 1,2,4-trioxolanes occurs via the high-energy chair conformers shown above, wherein the peroxide bond is exposed for reaction with Fe(II). Historically, drug discovery efforts in this class have focused solely on cis-R4 substitution (shown in blue) as in the “OZ compounds” arterolane and artefenomel (above, left). Our lab is exploring trans-R3 substitution (shown in red), which offers more options for the control of conformational equilibria, and thus Fe(II) reactivity.

Targeted drug delivery

Inspired by the above-referenced antimalarial endoperoxides and their remarkable pharmacology, our lab has pioneered the concept of Fe(II)-dependent delivery of therapeutics across therapeutic areas. The premise underlying this work is that normal cells and tissues have only a nominal (mid-nM) concentration of free (“labile”) ferrous iron, whereas significant concentrations of this redox–active and toxic species is present in certain diseased cells and tissues. For example, cystic fibrosis patients have been found to possess high micromolar concentrations of free Fe(II) in their sputum as a result of lung colonization by Pseudomonas aeruginosa biofilms. This can be contrasted with the near absence of free Fe(II) in human plasma (estimated at < 10-16 M). Clinical and epidemiological evidence from breast and prostate cancer patients indicates that iron-avid tumors are more aggressive, with poorer outcomes for cancer patients.

To detect and target drugs to ferrous iron avid tumors and tissues, we designed the scaffold TRX-C as a small molecule platform for Fe(II)-dependent delivery of diverse small molecule payloads (both therapeutics and reporters). We have now successfully employed this chemistry to invent useful new in vitro and in vivo probes of Fe(II), and to deliver therapeutics in animal models of malaria, cancer, and bacterial infection. Our current efforts in this area are focused on the more selective delivery of antibiotic payloads and on the application of the TRX scaffold as notional “safety­–catch” linkers for antibody-drug conjugates (ADCs).


Ferrous iron-dependent delivery of the antimalarial mefloquine: The TRX-C conjugate of the antimalarial mefloquine was found to be equally effective as mefloquine in mouse models of malaria, while limiting mefloquine exposure in the brain. This therapeutic approach might therefore be employed to mitigate the serious CNS side effects of this otherwise effective antimalarial drug. Image includes a derivative of human brain by Hugh Guiney (Creative Commons Attribution-Share Alike 3.0 Unported license).

Imaging reagents

To enable basic research into iron metabolism and distribution in vivo, and as prototypical diagnostic reagents for TRX drug conjugates, we are developing first-in-class reagents to image ferrous iron in experimental animals and humans using bioluminescent and PET imaging methods. These tools are being constructed with the same TRX-C moiety employed in our therapeutic conjugates, or by the introduction of PET radionuclides into Fe(II)-reactive endoperoxides with optimized in vivo properties.


Imaging ferrous iron: Left: the new reagent ICL-1 comprising D-aminoluciferin caged with the TRX-C moiety allows imaging of mobilized ferrous iron in live mice infected with the Gm-negative pathogen A. baumannii [Aron et al., 2017]. Right: a prototypical TRX-based PET reagent detects pools of labile Fe(II) in live mice [with Evans Lab, UCSF].

Selected recent publications

  1. Spangler B, Kline T, Hanson J, Li X, Zhou S, Wells JA, Sato AK, Renslo AR. Toward a Ferrous-Iron Cleavable Linker for Antibody-Drug Conjugates. Mol Pharm. 2018,15, 2054. PMID: 29569925

  2. Blank BR, Gut J, Rosenthal PJ, Renslo AR. Enantioselective Synthesis and in Vivo Evaluation of Regioisomeric Analogues of the Antimalarial Arterolane. J. Med. Chem. 2017, 60, 6400. PMCID: PMC5535261

  3. Aron AT, Heffern MC, Lonergan ZR, Vander Wal MN, Blank BR, Spangler B, Zhang Y, Park HM, Stahl A, Renslo AR, Skaar EP, Chang CJ. In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection. Proc Natl Acad Sci U S A. 2017, 114, 12669.

  4. Spangler B, Fontaine SD, Shi, Y, Sambucetti L, Mattis AN, Hann B, Wells JA, Renslo AR. A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron is Effective in Multiple Preclinical Cancer Models. J. Med. Chem. 2016, 59, 11161-11170. PMCID: PMC5184369

  5. Spangler B, Morgan, CW, Fontaine SD, Vander Wal MN, Chang CJ, Wells JA, Renslo AR. A reactivity-based probe of the intracellular labile ferrous iron pool. Nat. Chem. Biol. 2016, 12, 680-685. PMCID: PMC4990480

  6. Lauterwasser EMW, Fontaine SD, Li H, Gut J, Katneni K, Charman SA, Rosenthal PJ, Bogyo M, Renslo AR. Trioxolane-Mediated Delivery of Mefloquine Limits Brain Exposure in a Mouse Model of Malaria. ACS Med. Chem. 2015, 6, 1145-1149. PMCID: PMC4645246.

Novel neuropharmacology

Among the earliest and most successful drugs of the 20th century were molecules targeting G-protein coupled receptors (GPCR), cell-surface proteins that bind endogenous small-molecule ligands (e.g., histamine). Our lab is working to define new types of small molecule pharmacology for well-validated brain targets that have no known endogenous small molecule ligands.

Integrated Stress Response Inhibitors

In collaboration with the laboratories of Michelle Arkin, PhD, and Peter Walter, PhD, we identified and chemically optimized a novel class of inhibitors of the cellular stress response. These compounds, dubbed “ISRIB” for Integrated Stress Response InhiBitors, act by the unprecedented molecular mechanism of activating the guanine exchange activity of eIF2B, making stressed cells insensitive to the translation repression conferred by eIF2a phosphorylation. As such, ISRIB analogs act downstream of the eIF2a kinases (e.g., PERK, HRI, etc.) that have been the target of more traditional drug discovery efforts. In animal models, ISRIB has shown remarkable effects ranging from enhancement of memory consolidation to neuroprotection in prion disease and recovery from traumatic brain injury. Further development of these compounds towards the clinic is being undertaken by biopharmaceutical company Calico.

molecular diagram and visualization

ISRIB structure and binding to eIF2B: Left: structure-activity relationship studies of ISRIB in our lab suggested a symmetrical binding site. Right: the recent cryo-EM structure of human eIF2B with ISRIB bound at the interface of β and δ subunits confirms the symmetrical nature of the ISRIB binding site. Watch an animated visualization: eIF2B-ISRIB cryo-EM structure.

Modulating ion-channel function

Potassium two-pore (K2P) channels are voltage-gated ion channels that produce “leak” currents to control electrical excitability in the brain, cardiovascular system, and somatosensory system. The TREK subfamily of K2Ps is important in ischemia, neuroprotection, pain, depression, and anesthetic responses. Unfortunately, chemical validation of TREK channels as drug targets has been hindered by a dearth of small molecule ligands that modulate TREK function. Working with the Minor Lab at UCSF, we are using a combination of X-ray crystallography, protein engineering, and synthetic/medicinal chemistry to better understand the mechanism of known TREK modulators (e.g., BL-1249) and to discover and characterize new small molecules and their cognate modulatory binding sites. To date, we have described two new classes of TREK activators (ML67-33 and ML335) that bind at distinct, previously unknown sites. Our current efforts are focused on turning these nascent small molecules into chemical biology tools and drug leads to study TREK function in cells, tissue slices, and whole animals.

molecular diagram

Defining and expanding K2P potassium channel pharmacology: Recent studies from the Minor Lab and the Renslo Lab have revealed the likely binding site of BL-1249 at the C-terminal tail [Pope et al., 2018] and identified a novel modulatory site adjacent to the selectivity filter that binds the activator ML-335 [Lolicato et al., 2017]. Previously, we described ML67-33, a selective activator of the C-type gate in K2P channels [Bagriantsev et al., 2013].

Antiprion small molecules

In collaboration with Stanley Prusiner and the UCSF Institute for Neurodegenerative Diseases, our lab helped identify and optimize a new class of antiprion small molecules (2-aminothiazoles or AMTs) that were among the first shown to significantly extend survival in prion-infected mice. Brain-penetrant AMTs such as SMDC-256082 (aka IND24) extend the lives of prion-infected mice by about 100 to 200 days when used therapeutically and by more than 350 days when used prophylactically. Moreover, extensive studies of these compounds in mice have revealed a novel form of drug resistance arising from “conformational mutagenesis” of prion strains. While the molecular target of these molecules is yet to be firmly established, their strain-dependent effects and ability to select for drug-resistant “strains” (i.e., protein conformations) suggests they might bind and stabilize transiently formed conformers of the prion protein PrPC along the energy landscape of protein misfolding.

Aminothiazoles: Left: structure-activity profile of antiprion 2-aminothiazoles. Right: relationship between dose and survival index for IND24 in mice infected with RML or ME7 strain prions.

Selected recent publications

  1. Tsai JC, Miller-Vedam LE, Anand AA, Jaishankar P, Nguyen HC, Renslo AR, Frost A, Walter P. Structure of the Nucleotide Exchange Factor eIF2B Reveals Mechanism of Memory-Enhancing Molecule. Science, 2018, 359, 6383. PMID: 29599213

  2. Hearn BR, Jaishankar P, Sidrauski C, Tsai JC, Vedantham P, Fontaine SD, Walter P, Renslo AR. Structure-Activity Studies of Bis-O-Arylglycolamides: Inhibitors of the Integrated Stress Response. ChemMedChem. 2016, 11, 870-80. PMID: 26789650.

  3. Giles K, Berry DB, Condello C, Hawley RC, Gallardo-Godoy A, Bryant C, Oehler A, Elepano M, Bhardwaj S, Patel S, Silber BM, Guan S, DeArmond SJ, Renslo AR, Prusiner SB. Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains. J. Pharmacol. Exp. Ther. 2015, 355:2-12. PMCID: PMC4576665

  4. Ghaemmaghami S, Russo M, Renslo AR. Successes and challenges in phenotype-based lead discovery for prion diseases. J. Med. Chem., 2014, 57, 6919. PMCID: PMC4148153.

  5. Bagriantsev SN, Ang KH, Gallardo-Godoy A, Clark KA, Arkin MR, Renslo AR, Minor DL. A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K2P Channels. ACS. Chem. Biol. 2013, 8, 1841-2851. PMCID: PMC3747594

Fragment-based lead discovery

Our lab employs biophysical methods to identify fragments and eventually leads for venerable drug targets (e.g., β-lactamases), so-called “undruggable” targets (e.g., Ras), and protein-protein interfaces (e.g., pro-caspase 6, 14-3-3). Through the Small Molecule Discovery Center (SMDC) we perform fragment screens using surface plasmon resonance and MS-based disulfide-exchange screening (“tethering”). To enable the latter, our lab synthesized a unique screening library of about 1,800 disulfide fragments. This library has been screened against more than a dozen targets, yielding novel chemical probes/leads for kinases (e.g., allosteric modulators of PDK, with Jim Wells, PhD), Ras (with Frank McCormick, PhD, FRS, DSc (Hon) and Kevan M. Shokat, PhD), Caspase-6 and 14-3-3 (with Michelle Arkin, PhD), and dimeric herpes virus proteases (with Charles S. Craik, PhD). Our collaborative work on β-lactamases with the Chen Lab at University of Southern Florida has resulted in novel classes of reversible, non-covalent inhibitors of expanded spectrum β-lactamases and carbapenemases.

molecular diagram

Reversible inhibitors of CTX-M β-lactamase: Structural information about fragment binding to CTX-M binding mode of fragments (left and right) was used to generate more potent leads (center). Further optimization of these leads yielded the first non-covalent, reversible inhibitors of β-lactamase to exhibit activity in live bacteria.

Selected recent publications

  1. Pemberton OA, Zhang X, Nichols DA, DeFrees K, Jaishankar P, Bonnet R, Adams J, Shaw LN, Renslo AR, Chen Y. Antibacterial Spectrum of a Tetrazole-Based Reversible Inhibitor of Serine β-Lactamases. Antimicrob. Agents Chemother. 2018, 62, e02563-17. PMID: 29844038

  2. Gentile DR, Rathinaswamy MK, Jenkins ML, Moss SM, Siempelkamp BD, Renslo AR, Burke JE, Shokat KM. Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States. Cell Chem Biol. 2017 24, 1455. PMID: 29033317

  3. Murray J, Giannetti AM, Steffek M, Gibbons P, Hearn BR, Cohen F, Tam C, Pozniak C, Bravo B, Lewcock J, Jaishankar P, Ly CQ, Zhao X, Tang Y, Chugha P, Arkin MR, Flygare J, Renslo AR. Tailoring small molecules for an allosteric site on procaspase-6. 2014, ChemMedChem, 9(1), 73-77. PMID: 24259468

  4. Turner DM, Tom CT, Renslo AR. Simple Plate-Based, Parallel Synthesis of Disulfide Fragments using the CuAAC Click Reaction. ACS Comb Sci. 2014 Dec 8; 16(12):661-4. PMID: 25353066