Cell Chemical Biology

Ferroptosis is a non-apoptotic form of cell death resulting from the iron-dependent accumulation of lipid peroxides. Colorectal cancer (CRC) cells accumulate high levels of intracellular iron and reactive oxygen species (ROS) and are thus particularly sensitive to ferroptosis. The compound (S)-RSL3 ([1S,3R]-RSL3) is a commonly used ferroptosis inducing compound that is currently characterized as a selective inhibitor of the selenocysteine containing enzyme (selenoprotein) Gluathione Peroxidase 4 (GPx4), an enzyme that utilizes glutathione to directly detoxify lipid peroxides. However, through chemical controls utilizing the (R) stereoisomer of RSL3 ([1R,3R]-RSL3) that does not bind GPx4, combined with inducible genetic knockdowns of GPx4 in CRC cell lines, we revealed that GPx4 dependency does not always align with (S)-RSL3 sensitivity, questioning the current characterization of GPx4 as the central regulator of ferroptosis. Utilizing affinity pull-down mass spectrometry with chemically modified (S)-RSL3 probes we discovered that the effects of (S)-RSL3 extend far beyond GPx4 inhibition, revealing that (S)-RSL3 is a broad and non-selective inhibitor of selenoproteins. To further investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed additional chemical and genetic approaches. We found that the selenoprotein inhibitor auranofin, an FDA approved gold-salt, chemically induced oxidative cell death and ferroptosis in both in-vitro and in-vivo models of CRC. Consistent with these data, we found that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translation of selenoproteins, is essential for the in-vitro growth and xenograft survival of CRC cell lines. In summary, these findings recharacterize the mechanism of action of the most commonly used ferroptosis inducing molecule, (S)-RSL3, and reveal that broad inhibition of selenoproteins is a promising novel therapeutic angle for the treatment of CRC.

Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important innovation in the development of drugs against a range of pathogens and other pathologies. A broad spectrum of pharmacology is associated with chemically diverse BoB derivatives and includes multiple modes-of-action (MoA) and targets. However, a consensus MoA for BoBs targeting evolutionarily diverse protozoan pathogens has emerged with the identification of CPSF3/CPSF73 in the CPSF complex in both apicomplexan and kinetoplastida parasites. Here we establish a functional connection between protein sumoylation and the boron-heterocyclic scaffold shared by all BoBs using comprehensive genetic screens in Trypanosoma brucei. There is a rapid temporal and spatial shift in global protein sumoylation following BoB exposure and members of the CPSF complex are specifically destabilised in a SUMO and proteosome-dependent manner. Finally, we find rapid decrease in bulk mRNA levels, consistent with the role of CPSF3 in mRNA maturation. We propose that a combination of direct inhibition coupled with targeted degradation of CPSF3 underpins the specificity of BoBs against trypanosomatids.

Certain arylsulfonamides (ArSulfs) induce an interaction between the E3 ligase substrate adaptor DCAF15 and the critical splicing factor RBM39, ultimately causing its degradation. Although molecules like the ArSulfs, which interfere with splicing decisions, are exciting potential medicines, the molecular glue mechanism of RBM39 degradation introduces complex pleiotropic effects that are difficult to untangle. For example, DCAF15 inhibition, RBM39 degradation, and the downstream proteome effects of splicing changes will all cause different yet overlaid effects. As such the precise cell-killing mechanism by RBM39 loss is largely unknown. By overlaying transcriptome and proteome datasets, we distinguish transcriptional from post-transcriptional effects, pinpointing those proteins most impacted by splicing changes. Our proteomic profiling of several ArSulfs suggests a selective DCAF15/ArylSulf/RBM39RRM2 interaction with a narrow degradation profile. We identify two mitotic kinesin motor proteins that are aberrantly spliced upon RBM39 degradation, and we demonstrate that these are likely contributors to the antiproliferative activity of ArSulfs.

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the {beta}5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of {beta}5 plus either {beta}1 or {beta}2 results in improved potency.

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

Targeted protein degradation (TPD) with Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin-proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D--EN67--that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D-dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.

Proteolysis-targeting chimeras (PROTACs) co-op the ubiquitin system for targeted protein degradation, creating opportunities to interrogate cellular functions of proteins through "chemical knockdown". However, matched pairs of protein degraders and inhibitors, that possess high specificity and chemical complementarity, for individual components of the ubiquitin system have remained scarce. This includes reagents to modulate activity and abundance of deubiquitinases (DUBs), which critically regulate ubiquitin-mediated signaling. Here, using an integrated chemical biology approach, we explored the cellular function of the DUB USP7 as a case study comparing inhibition and degradation of this DUB in melanoma and pancreatic cancer cells. Through the synthesis of a degrader library, we identified potent USP7 PROTACs for each cancer type, established BRET-based ternary complex formation and quantified degradation efficiency. USP7 degraders and their cognate inhibitor were subsequently employed to characterize treatment-induced phenotypic alterations. Proteomic and cellular analyses revealed that highly specific degradation of USP7 modulated both shared and distinct protein sets across cancer cell types, without impacting cell growth. Notably, cellular responses to USP7 degradation differed markedly from those to USP7 inhibition. Moreover, our data uncovered broad proteomic and metabolic changes induced by prolonged USP7 inhibitor treatment. Collectively, our work provides a chemical toolbox of comprehensively characterized reagents to distinguish on-target phenotypes which will aid the understanding of the role of USP7 in malignant diseases. More broadly, our data emphasize the importance of increased specificity via PROTAC-mediated degradation and the potential of this modality to distinguish catalytic from non-catalytic as well as cell-line specific functions of DUBs.

Benzoxaboroles are emerging as promising treatments against a broad range of protozoan parasites, including Trypanosoma, Leishmania, Plasmodium, Toxoplasma, and Cryptosporidium. We previously demonstrated that the benzoxaborole compound AN3661 inhibits the endonuclease activity of CPSF3 in Cryptosporidium hominis, likely by disrupting pre-mRNA processing, which in turn limits parasite growth. In this study, we further explored its mode of action and found that a specific mutation (Y385N) in CPSF3 of C. parvum confers strong resistance to AN3661. Interestingly, this mutation does not affect the parasites sensitivity to two other benzoxaboroles, AN13762 and AN7973, which are also thought to target CpCPSF3. All three compounds interfered with mRNA processing in C. parvum, consistent with inhibition of CPSF3 complex activity, and showed parasiticidal activity, especially during the late stages of merogony, blocking parasite egress. Two of them also impaired gamogony. In T. gondii, AN7973 remained effective against several strains carrying mutations within TgCPSF3 that confer resistance to AN3661 and AN13762, suggesting that it might represent an alternative chemotype targeting CPSF3 with the potential to overcome resistance. Notably, AN7973 successfully controlled severe infection in susceptible mice challenged with the AN3661-resistant C. parvum strain carrying the CpCPSF3Y385N mutation. Finally, we extended the known antiparasitic spectrum of AN3661 and AN7973 to include Eimeria tenella and Giardia duodenalis, two important pathogens in veterinary and human health. Altogether, our findings refine the understanding of CPSF3-targeting benzoxaboroles, identify alternative chemotypes with the potential to bypass resistance, and support their potential use in combination therapies to delay or prevent the emergence of drug resistance. Author summaryProtozoa are single-celled parasites that cause significant morbidity and mortality worldwide, affecting both humans and animals. The development of new targeted therapies with highly selective compounds requires a detailed understanding of their mode of action and precise interactions with parasite targets. Benzoxaboroles are a potent class of molecules active against Cryptosporidium, with compound AN3661 known to inhibit the endonuclease activity of the Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) in Cryptosporidium hominis. Here, we show that this inhibitory effect is critical during both asexual and sexual developmental stages and that tyrosine at position 385 of C. parvum CPSF3 plays a key role in its inhibition. A mutation at this site confers resistance to AN3661 both in vitro and in vivo, but not to two other benzoxaboroles, AN13762 and AN7973. Notably, all three compounds disrupted pre-mRNA processing in Cryptosporidium, consistent with inhibition of the CPSF3 complex. Using Toxoplasma, a related protozoan that allows more efficient genetic manipulation, we found that none of the six mutations conferring resistance to compounds AN3661 and AN13762 conferred resistance to compound AN7973. AN7973, which strongly inhibits both C. parvum and T. gondii, may therefore represent an alternative chemotype targeting CPSF3. Furthermore, we demonstrated that the antiparasitic spectrum of AN3661and AN7973 extends to include E. tenella and G. duodenalis, two important pathogens in veterinary and human health. Altogether, our results refine the understanding of three CPSF3-targeting benzoxaboroles in Cryptosporidium and identify compound AN7973 as capable of overcoming resistance to AN3661, thereby supporting the rationale for combination therapies to prevent the emergence of drug resistance.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

Release (egress) of malaria parasites from host red blood cells (RBC) is a protease-dependent process involving breakdown of the RBC cytoskeleton by a parasite cysteine protease-like protein called SERA6. In the penultimate step of the egress cascade, SERA6 undergoes autoproteolytic maturation triggered upon cleavage by a serine protease called SUB1 and requiring interactions between SERA6 and fragments of another parasite protein called MSA180. Egress can be blocked by treatment of intraerythrocytic parasites with small molecules that prevent the autocatalytic SERA6 maturation step, suggesting that SERA6 is a druggable target. Here we describe the development of a cell-free in vitro system that recapitulates SERA6 maturation. We use the assay to confirm the strict requirement for MSA180 in SERA6 maturation by SUB1 and to show that these 3 components are sufficient for SERA6 maturation. Using a synthetic peptide substrate based on a predicted autocatalytic cleavage site we demonstrate that the fully mature SERA6 is an active proteolytic enzyme and we validate improved small molecule inhibitors of SERA6. Our lead inhibitory compound efficiently blocks egress of asexual blood stage parasites, confirming SERA6 as a new potential antimalarial drug target.

L-Tryptophan (Trp) is an essential amino acid, catabolised through the kynurenine pathway, which is mediated by the enzymes indoleamine-2,3-dioxygenase 1 (IDO1), IDO2, or Trp-2,3-deoxygenase (TDO). Therapeutic targeting of Trp metabolism could be relevant to several pathologies. In cancer, IDO1 acts as an immune checkpoint suppressing effector T cell function. Yet, direct inhibition of IDO1 has had limited success in clinical trials. Therefore, alternative approaches to Trp metabolism therapeutic targeting are needed. We screened a library of 597 natural products (NPs) or NP derivatives for their effect on kynurenine production in triple negative breast cancer cells. This revealed 24 candidate inhibitors of kynurenine production. Amongst them, artemether, a member of the artemisinin family of anti-malarial drugs, suppressed kynurenine production, likely via an endoperoxide bridge-dependent mechanism. The Euphorbia factor L9 (EFL9) inhibited kynurenine production likely via a C7-benzoylation-dependent mechanism. Neither artemether nor EFL9 affected JAK/STAT signaling or IDO1 levels. Targeted metabolomics analyses demonstrated that artemether suppressed kynurenine production through heme sequestration, a mechanism that would affect all members of the IDO/TDO family of metalloenzymes. EFL9 affected purine and amino acid metabolism and the cellular redox balance. Comparisons to the effects of ouabain, a NP regulator of IDO1 levels, and Linrodostat, a clinically used small molecule IDO1 inhibitor, revealed distinct metabolic profiles, with ouabain and EFL9 showing the largest overlap. Importantly, the kynurenine-suppressing activity of artemether and EFL9 is not cancer cell-specific. Overall, our findings set the foundation for the use of derivatives of artemether or EFL9 as novel Trp metabolism-targeting therapeutics.

With resistance to most antimalarials increasing, it is imperative that new antimalarial drugs are developed to replace or complement front-line artemisinin therapies. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited ring development of newly invaded merozoites. Here, we selected parasites resistant to M-833 and identified independent mutations arising in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introduction of the identified PfSTART1 mutations into wildtype parasites reproduced resistance to both M-833 and highly potent analogues, confirming PfSTART1 mutations were sufficient to confer resistance. The analogues bound to recombinant PfSTART1 with nanomolar affinity. We also demonstrated selective PfSTART1 engagement by the analogues using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assay for the first time in Plasmodium. Imaging of newly invaded merozoites showed the inhibitors prevented the conversion into larger amoeboid ring-stage parasites potentially through the inhibition of phospholipid transfer from the parasite to the encasing parasitophorous vacuole membrane (PVM) and/or within the parasite. We show that these PfSTART1 inhibitors also block transmission. With multiple stages of the parasites lifecycle being targeted by PfSTART1 inhibitors, this protein therefore represents a novel drug target with a new mechanism of action.

Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective small molecule modulators can be challenging due to structural similarities in the sterol binding domains. We report the discovery of highly potent and selective inhibitors of oxysterol binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol binding proteins by targeted degradation, led to a significant reduction in Golgi-associated proteins. The degradation was found to occur at lysosomes, concomitant with changes in general protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target any other sterol transporters tested, we advocate their use as chemical tools to study OSBP function and therapeutic relevance.

Reversible inactivation of protein tyrosine phosphatases by reactive oxygen species (ROS) is essential to the phosphorylation of growth factor receptors. An important outcome of the inactivation of protein tyrosine phosphatase 1B (PTP1B) by ROS involves the conformational change of its phosphotyrosine binding loop which adopts a solvent exposed position in its oxidized form. We previously demonstrated that 14-3-3{zeta} binds to the phosphotyrosine binding loop of the oxidized form of PTP1B. Using a rational approach, we developed a unique protein-protein interaction (PPI) inhibitor peptide derived from the phosphotyrosine binding loop of PTP1B designed to disrupt the interaction between PTP1B and the 14-3-3{zeta}-complex. Exploiting this cell-permeable peptide, we showed decreased association between PTP1B and the 14-3-3{zeta}-complex in cells treated with epidermal growth factor (EGF). We also demonstrated that preventing the association of this 14-3-3{zeta}-complex to PTP1B deterred oxidation and inactivation of PTP1B following EGF receptor (EGFR) activation and generation of ROS. Treating cells with our PPI inhibitor decreased EGFR phosphorylation on PTP1B-specific sites. Furthermore, treating EGFR-driven epidermal cancer cells with our PPI inhibitor also significantly inhibited colony formation and cell viability, consitent with increased activation of PTP1B. These data highlight the ability of PTP1B to downregulate critical signaling pathways in cancer when activated using peptide drugs such as our protein-protein interaction inhibitor. We anticipate that preventing or destabilizing the reversible oxidation of other members of the protein tyrosine phosphatase superfamily using PPI inhibitors may offer a foundation for a broad therapeutic approach to rectify dysregulated signaling pathways in vivo. Significance StatementLimited understanding of redox mechanisms regulating PTP catalytic activity is a major knowledge gap that has hampered our efforts to develop activation strategies. In its reversibly oxidized and inactivated form, conformational changes of PTP1B influence its association with regulatory proteins. We demonstrate that designing a cell-permeable peptide based on a loop of PTP1B that becomes exposed during oxidation can block its interaction with the 14-3-3{zeta}-multiprotein complex and activate the phosphatase. Moreover, activating PTP1B using our protein-protein interaction inhibitor peptide decreases the phosphorylation of its substrate EGFR and decreases the effectiveness of cancer cells to form colonies. This study provides important insights into the therapeutic potential of protein-protein interaction inhibitors that regulate the redox cycle of PTPs to reestablish physiological signaling.

Puromycin is an amino nucleoside that inhibits protein synthesis by interrupting elongation of nascent peptide chains. It is a commonly used selection antibiotic in molecular biology research via engineered expression of a puromycin resistance transgene. The enzyme puromycin acetyl transferase (pac) or PuroR inactivates puromycin by N-acetylating its reactive amino group. Puromycin acetylation by pac requires the central metabolite and acetyl group donor acetyl-CoA as a substrate. We found that puromycin treatment exacerbates sensitivity of cancer cells to knockdown of pantothenate kinases, the proteins that catalyze the rate-limiting step of de novo coenzyme A production in cells. Mechanistically, we found that ablation of PANKs together with puromycin depletes acetyl-CoA levels, in a manner modulated by the dose of puromycin. Our findings provide a note of caution and context in the use of puromycin for metabolism research in that interference with the major acyl donor used for inactivating biotransformation may exacerbate toxicity under selection. Broadly, our findings also invite studies to explore how targeting CoA and acetyl-CoA synthesis may be exploited to enhance cytotoxic effects of cancer drugs that undergo acetylation.

The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, being particularly problematic in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and the overall dearth of new drug targets for Schistosoma. Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for activity against P. falciparum. We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. Evolution of resistance using a mutator P. falciparum line revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy-number amplification of a phospholipid-translocating ATPase, pfatp2, a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy-number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum.

MicroRNA families are pervasive in the human transcriptome, but specific targeting of individual members is a challenge because of sequence homology. Many of the secondary structures of the precursors to these miRs (pre-miRs), however, are quite different. Here, we demonstrate both in vitro and in cellulis that design of structure-specific small molecules can inhibit specific miR family members to modulate a disease pathway. In particular, the miR-200 family consists five miRs, miR-200a, −200b, −200c, −141, and - 429, and is associated with Type II Diabetes (T2D). We designed a small molecule that potently and selectively targets pre-miR-200c’s structure. The compound reverses a pro-apoptotic effect in a pancreatic β-cell model. In contrast, oligonucleotides targeting the RNA’s sequence inhibit all family members. Global proteomics analysis further demonstrates selectivity for miR-200c. Collectively, these studies establish that miR-200c plays an important role in T2D and that small molecules targeting RNA structure can be an important complement to oligonucleotides targeting sequence.Significance Statement The most common way to develop medicines targeting RNA is by using oligonucleotides that target its sequence by using base pairing. Some RNAs, however, have similar sequences and thus are impossible to target selectively by using oligonucleotides. Here, we show that a class of RNAs that have similar sequences emerge from precursors that have very different structures. Exploiting these structural differences afforded a selective compound. In particular, the selective small molecule targets a member of the microRNA (miR)-200 family, the overexpression of which is linked to diabetes and pancreatic cell death. Selective inhibition of family member miR-200c alleviates pancreatic cell death, and thus the small molecule provides a path to the treatment of diabetes.Competing Interest StatementMDD is a founder of expansion therapeuticsView Full Text

Artemisinin resistant Plasmodium falciparum (Pf) is spreading despite combination chemotherapy (ACT). Here we report the design of artezomibs, single-molecule hybrids of an artemisinin and a Pf-selective proteasome inhibitor. Artezomibs exert a novel mode of action inside the malaria parasites. The artemisinin component covalently modifies parasite proteins, which become substrates of the Pf proteasome. The proteasomal degradation products that bear the proteasome inhibitor component of the hybrid then inhibit Pf proteasomes, including those with mutations that reduce binding affinity of the proteasome inhibitor component on its own. We demonstrated that artezomibs circumvent both artemisinin resistance conferred by Kelch13 polymorphism and resistance to the proteasome inhibitor associated with mutations in Pf proteasomes. This mode of action may enable the use of a single molecule with one pharmacokinetic profile to prevent the emergence of resistance.

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

Enhancing proteasome activity is a potential new therapeutic strategy to prevent the accumulation of aberrant high levels of protein that drive the pathogenesis of many diseases. Herein, we examine the use of small molecules to activate the 20S proteasome to reduce aberrant signaling by the undruggable oncoprotein c-MYC, to treat c-MYC driven oncogenesis. Overexpression of c-MYC is found in more than 50% of all human cancer but remains undruggable because of its highly dynamic intrinsically disordered 3-D conformation, which renders traditional therapeutic strategies largely ineffective. We demonstrate herein that small molecule activation of the 20S proteasome targets dysregulated intrinsically disordered proteins (IDPs), including c-MYC, and reduces cancer growth in vitro and in vivo models of multiple myeloma, and is even effective in bortezomib resistant cells and unresponsive patient samples. Genomic analysis of various cancer pathways showed that proteasome activation results in downregulation of many c-MYC target genes. Moreover, proteasome enhancement was well tolerated in mice and dogs. These data support the therapeutic potential of 20S proteasome activation in targeting IDP-driven proteotoxic disorders, including cancer, and demonstrate that this new therapeutic strategy is well tolerated in vivo.