Michael-addition of a thiol to a maleimide is commonly used for bioconjugation of drugs to macromolecules. Indeed, both current FDA-approved antibody-drug conjugates-Brentuximab vedotin and Trastuzumab emtansine-and one approved PEGylated conjugate-Cimzia-contain a thiol-maleimide adduct. However, the ultimate in vivo fate of such adducts is to undergo disruptive cleavage by thiol exchange or stabilizing ring opening. Therapeutic efficacy of a conjugate can be compromised by thiol exchange and the released drug may show toxicities. However, if the succinimide moiety of a maleimide-thiol conjugate is hydrolyzed, the ring-opened product is stabilized toward cleavage. We determined rates of ring-opening hydrolysis and thiol exchange of a series of N-substituted succinimide thioethers formed by maleimide-thiol conjugation. Ring-opening of conjugates prepared with commonly used maleimides were too slow to serve as prevention against thiol exchange. However, ring-opening rates are greatly accelerated by electron withdrawing N-substituents, and ring-opened products have half-lives of over two years. Thus, conjugates made with electron-withdrawing maleimides may be purposefully hydrolyzed to their ring-opened counterparts in vitro to ensure in vivo stability.

Improved methods for studying intracellular reactive iron(II) are of significant interest for studies of iron metabolism and disease relevant changes in iron homeostasis. Here we describe a highly-selective reactivity-based probe in which Fenton-type reaction with intracellular labile iron(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using the high-content, plate-based immunofluorescence assay described. Using this new probe and screening approach, we detected alteration of cellular labile iron(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. Finally, we utilized this new tool to demonstrate the presence of augmented labile iron(II) pools in cancer cells as compared to non-tumorigenic cells.

Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

PARP inhibitors are approved for treatment of cancers with BRCA1 or BRCA2 defects. In this study, we prepared and characterized a very long-acting PARP inhibitor. Synthesis of a macromolecular prodrug of talazoparib (TLZ) was achieved by covalent conjugation to a PEG40kDa carrier via a β-eliminative releasable linker. A single injection of the PEG∼TLZ conjugate was as effective as ∼30 daily oral doses of TLZ in growth suppression of homologous recombination-defective tumors in mouse xenografts. These included the KT-10 Wilms' tumor with a PALB2 mutation, the BRCA1-deficient MX-1 triple-negative breast cancer, and the BRCA2-deficient DLD-1 colon cancer; the prodrug did not inhibit an isogenic DLD-1 tumor with wild-type BRCA2. Although the half-life of PEG∼TLZ and released TLZ in the mouse was only ∼1 day, the exposure of released TLZ from a single safe, effective dose of the prodrug exceeded that of oral TLZ given daily over one month. μPET/CT imaging showed high uptake and prolonged retention of an 89Zr-labeled surrogate of PEG∼TLZ in the MX-1 BRCA1-deficient tumor. These data suggest that the long-lasting antitumor effect of the prodrug is due to a combination of its long t1/2, the high exposure of TLZ released from the prodrug, increased tumor sensitivity upon continued exposure, and tumor accumulation. Using pharmacokinetic parameters of TLZ in humans, we designed a long-acting PEG∼TLZ for humans that may be superior in efficacy to daily oral TLZ and would be useful for treatment of PARP inhibitor-sensitive cancers in which oral medications are not tolerated. Significance: These findings demonstrate that a single injection of a long-acting prodrug of the PARP inhibitor talazoparib in murine xenografts provides tumor suppression equivalent to a month of daily dosing of talazoparib.

Ferrous iron-promoted reduction of a hindered peroxide bond underlies the antimalarial action of the 1,2,4-trioxane artemisinin and the 1,2,4-trioxolane arterolane. In appropriately designed systems, a 1,2,4-trioxolane ring can serve as a trigger to realize ferrous iron-dependent and parasite-selective drug delivery, both in vitro and in vivo. A stereocontrolled, expeditious (three steps), and efficient (67–71% overall yield) synthesis of 1,2,4-trioxolanes possessing the requisite 3″ substitution pattern that enables ferrous iron-dependent drug delivery is reported. The key synthetic step involves a diastereoselective Griesbaum co-ozonolysis reaction to afford primarily products with a trans relationship between the 3″ substituent and the peroxide bridge, as confirmed by X-ray structural analysis of a 3″-substituted 4-nitrobenzoate analogue.

The goal was to develop and characterize a companion diagnostic for the releasable PEG 40kDa $SN-38 oncology drug, PLX038, that would identify tumors susceptible to high accumulation of PLX038. PEG conjugates of the zirconium ligand desferroxamine B (DFB) of similar size and charge to PLX038 were prepared that contained one or four DFB, as well as one that contained three SN-38 moieties and one DFB. Uptake and associated kinetic parameters of the 89 Zr-labeled nanocarriers were determined in tumor and normal tissues in mice using mPET/CT imaging. The data were fit to physiologically based pharmacokinetic models to simulate the mass-time profiles of distribution of conjugates in the tissues of interest. The timeactivity curves for normal tissues showed high levels at the earliest time of measurement due to vascularization, followed by a monophasic loss. In tumors, levels were initially lower than in normal tissues but increased to 9% to 14% of injected dose over several days. The efflux half-life in tumors was very long, approximately 400 hours, and tumor levels remained at about 10% injected dose 9 days after injection. Compared with diagnostic liposomes, the PEG nanocarriers have a longer serum halflife, are retained in tumors at higher levels, remain there longer, and afford higher tumor exposure. The small PEG 40kDa nanocarriers studied here show properties for passive targeting of tumors that are superior than most nanoparticles and might be effective probes to identify tumors susceptible to similar size therapeutic nanocarriers such as PLX038.

The antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane-like 1,2,4-trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared to our earlier drug delivery scaffolds, these new arterolane-inspired systems are of significantly reduced molecular weight and possess superior metabolic stability. We also demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live P. falciparum parasites.