Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles.

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.

Structure-activity relationships of GPX4 inhibitor warheads.

Direct inhibition of GPX4 requires covalent modification of the active-site selenocysteine. While phenotypic screening has revealed that activated alkyl chlorides and masked nitrile oxides can inhibit GPX4 covalently, a systematic assessment of potential electrophilic warheads with the capacity to inhibit cellular GPX4 has been lacking. Here, we survey more than 25 electrophilic warheads across several distinct GPX4-targeting scaffolds. We find that electrophiles with attenuated reactivity compared to chloroacetamides are unable to inhibit GPX4 despite the expected nucleophilicity of the selenocysteine residue. However, highly reactive propiolamides we uncover in this study can substitute for chloroacetamide and nitroisoxazole warheads in GPX4 inhibitors. Our observations suggest that electrophile masking strategies, including those we describe for propiolamide- and nitrile-oxide-based warheads, may be promising for the development of improved covalent GPX4 inhibitors.

Lithium bis-catechol borate as an effective reductive quencher in photoredox catalysis.

The use of lithium bis-catechol borate (LiB(cat)2) as a reductive quencher for the photoredox mediated intermolecular C-H functionalization of various heteroaromatics with bromopyrroloindolines is described. LiB(cat)2 offers a financial benefit over state-of-the-art quenchers currently in use while eliminating the side reactions that typically plague these couplings. The advantage of this methodology is highlighted by the synthesis of C3-C2' (-) gliocladin C. Furthermore, additional examples of reactivity with various bromopyrroloindolines sets the stage for expedient routes towards other pharmaceutically active hexahydropyrroloindoline alkaloids and their analogues.

Formation and Trapping of Azafulvene Intermediates Derived from Manganese-Mediated Oxidative Malonate Coupling.

The one-pot, three-component, coupling reaction of indoles/pyrroles, dimethyl malonate, and acetic acid was performed using Mn(III) acetate as an oxidant. In the presence of Mn(OAc)3, indole-2, and indole-3-carbonyl compounds were alkylated at the 3- and 2- positions, respectively, with subsequent oxidation and nucleophilic capture occurring at the newly formed benzylic carbon. In contrast, oxidation of 2- and 3-indole carboxylic acids afforded the corresponding 2-oxindol-3-ylidenes and 3-oxindol-2-ylidenes. The reaction conditions, scope, and mechanism are discussed herein.

Validation of ferroptosis in canine cancer cells to enable comparative oncology and translational medicine.

Ferroptosis is a cell death mechanism that has attracted significant attention as a potential basis for the development of new cancer therapies. Validation of ferroptosis biology in species commonly used in translation and pre-clinical development is a necessary foundation for enabling the advancement of such ferroptosis modulating drugs. Here, we demonstrate that canine cancer cells exhibit sensitivity to a wide range of ferroptosis-inducing perturbations in a manner indistinguishable from human cancer cells, and recapitulate characteristic patterns of ferroptotic response across tumor types seen in the human setting. The foundation provided herein establishes the dog as a relevant efficacy and toxicology model for ferroptosis and creates new opportunities to leverage the canine comparative oncology paradigm to accelerate the development of ferroptosis-inducing drugs for human cancer patients.

Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162.

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Šresolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

Biomimetic transannular oxa-conjugate addition approach to the 2,6-disubstituted dihydropyran of laulimalide yields an unprecedented transannular oxetane.

2,6-Disubstituted dihydropyrans are a common feature in many bioactive polyketides, including the anticancer marine polyketide laulimalide. While much of the uncharacterized biosynthetic pathway for laulimalide can be confidently postulated, the biosynthetic origins of the trans 2,6-disubstituted dihydropyran cannot. We hypothesize that a transannular oxa-conjugate addition in a macrocyclic laulimalide precursor could be the origin of the 2,6-dihydropyran. To test this hypothesis, we constructed a model containing the key functional groups for oxa-conjugate addition-mediated dihydropyran formation. Under acid-mediated conditions, the model under went regiospecific oxa-conjugate addition producing a stable trans oxetane as the only regioisomer. The desired, more stable dihydropyran was not detected. This unprecedented regiospecificity is unexpected due to the ring strain of the oxetane and the anticipated facile ring opening retro-oxa-conjugate addition. The oxetane is stable to acid and basic conditions, as are a number of literature acyclic oxetanes that could undergo similar retro-oxa-conjugate addition. While the source of the oxetane kinetic stability is yet to be characterized, it may enable general oxetane construction via oxa-conjugate addition. The more stable dihydropyran regioisomer could not be generated due to poor geometrical orbital alignment and hard-soft incompatibility between the hard oxygen nucleophile and the soft activated polyenoate electrophile. These factors disfavor the breaking of conjugation by oxa-conjugate addition. Based on these results we propose that dihydropyran formation does not occur on completed polyketide macrocycles as we had proposed but rather during polyketide biosynthesis on the growing polyketide chain.

Genomic Activation of PPARG Reveals a Candidate Therapeutic Axis in Bladder Cancer.

The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987-98. ©2017 AACR.

Efficient Routes to a Diverse Array of Amino Alcohol-Derived Chiral Fragments.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1-5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

Functionally diverse nucleophilic trapping of iminium intermediates generated utilizing visible light.

Our previous studies into visible-light-mediated aza-Henry reactions demonstrated that molecular oxygen played a vital role in catalyst turnover as well as the production of base to facilitate the nucleophilic addition of nitroalkanes. Herein, improved conditions for the generation of iminium ions from tetrahydroisoquinolines that allow for versatile nucleophilic trapping are reported. The new conditions provide access to a diverse range of functionality under mild, anaerobic reaction conditions as well as mechanistic insights into the photoredox cycle.

Visible light-mediated intermolecular C-H functionalization of electron-rich heterocycles with malonates.

The photoredox-mediated direct intermolecular C-H functionalization of substituted indoles, pyrroles, and furans with diethyl bromomalonate is described, utilizing the visible light-induced reductive quenching pathway of Ru(bpy)(3)Cl(2). An analysis of reductive quenchers and mechanistic considerations has led to an optimized protocol for the heteroaromatic alkylations, providing products in good yields and regioselectivities, as well as successfully eliminating previously observed competitive side reactions. This methodology is highlighted by its neutral conditions, activity at ambient temperatures, low catalyst loading, functional group tolerance, and chemoselectivity.