Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.

K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function.

The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated.

Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization-recommended drug, but concerns over drug resistance encourage the search for new drug leads.

Schistosome bloodflukes are complex trematodes responsible for 200 million cases of schistosomiasis worldwide. Their life cycle is characterized by a series of remarkable morphological and biochemical transitions between an invertebrate host, an aquatic environment, and a mammalian host. We report a global transcriptional analysis of how this parasite alters gene regulation to adapt to three distinct environments.

During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.

Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.

One inhibitor of the main SARS-CoV-2 protease has been approved recently by the FDA, yet it targets only SARS-CoV-2 main protease (Mpro). Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases involved in SARS-CoV-2 replication, including Mpro, SARS-CoV-2 papain-like protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by an idea to find a better alternative than disulfiram, an approved treatment for chronic alcoholism that is currently in phase 2 clinical trials against SARS-CoV-2. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage, improve the efficacy, and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, RI173, and RI172 were the most potent inhibitors in an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L, with IC50s of 300, 200, and 200 nM, which is about 5-, 19-, and 11-fold more potent than disulfiram, respectively. In addition, RI173 was tested against SARS-CoV-2 in a cell-based and toxicity assay and was shown to have a greater antiviral effect than disulfiram. The identified compounds demonstrated the promising potential of thiuram disulfide or dithiobis-(thioformate) as a reactive functional group in small molecules that could be further developed for treatment of the COVID-19 virus or related variants.

Cutaneous leishmaniasis (CL) is the most common disease form caused by a Leishmania parasite infection and considered a neglected tropical disease (NTD), affecting 700,000 to 1.2 million new cases per year in the world. Leishmania major is one of several different species of the Leishmania genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can provide knowledge of new targets for host-targeted drug development. In this study, tissue samples were collected from mice infected in the ear or footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including selected glycerophosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through host metabolism and may lead to new curative measures against infection with Leishmania.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

Redox cycling of iron powers various enzyme functions crucial for life, making the study of iron acquisition, storage, and disposition in the whole organism a worthy topic of inquiry. However, despite its important role in biology and disease, imaging iron in animals with oxidation-state specificity remains an outstanding problem in biology and medicine. Here we report a first-generation reactivity-based probe of labile ferrous iron suitable for positron emission tomography studies in live animals. The responses of this reagent to systemic changes in labile iron disposition were revealed using iron supplementation and sequestration treatments in mice, while the potential of this approach for in vivo imaging of cancer was demonstrated using genetically and pathologically diverse mouse models, including spontaneous tumors arising in a genetically engineered model of prostate cancer driven by loss of PTEN.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Safe and effective treatments for Chagas disease, a potentially fatal parasitic infection associated with cardiac and gastrointestinal pathology and caused by the kinetoplastid parasite Trypanosoma cruzi, have yet to be developed. Benznidazole and nifurtimox, which are currently the only available drugs against T. cruzi, are associated with severe adverse effects and questionable efficacy in the late stage of the disease. Natural products have proven to be a rich source of new chemotypes for other infectious agents. We utilized a microscopy-based high-throughput phenotypic screen to identify inhibitors of T. cruzi from a library of natural product samples obtained from fungi procured through a Citizen Science Soil Collection Program (https://whatsinyourbackyard.org/) and the Great Lakes (USA) benthic environment. We identified five leucinostatins (A, B, F, NPDG C, and NPDG D) as potent inhibitors of the intracellular amastigote form of T. cruzi. Leucinostatin B also showed in vivo suppression of T. cruzi in a mouse model of Chagas disease. Given prior reports that leucinostatins A and B have antiparasitic activity against the related kinetoplastid Trypanosoma brucei, our findings suggest a potential cross-trypanocidal compound class and provide a platform for the further chemical derivatization of a potent chemical scaffold against T. cruzi.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.