Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Quantitative self-assembly prediction yields targeted nanomedicines.

Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.

DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis.

Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1ß but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.

Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases.

Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ~20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome.

The danger signals that activate the NLRP1 inflammasome have not been established. Here, we report that the oxidized, but not the reduced, form of thioredoxin-1 (TRX1) binds to NLRP1. We found that oxidized TRX1 associates with the NACHT-LRR region of NLRP1 in an ATP-dependent process, forming a stable complex that restrains inflammasome activation. Consistent with these findings, patient-derived and ATPase-inactivating mutations in the NACHT-LRR region that cause hyperactive inflammasome formation interfere with TRX1 binding. Overall, this work strongly suggests that reductive stress, the cellular perturbation that will eliminate oxidized TRX1 and abrogate the TRX1-NLRP1 interaction, is a danger signal that activates the NLRP1 inflammasome.

DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes.

Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.

Caspase-1 interdomain linker cleavage is required for pyroptosis.

Pathogen-related signals induce a number of cytosolic pattern-recognition receptors (PRRs) to form canonical inflammasomes, which activate pro-caspase-1 and trigger pyroptotic cell death. All well-studied inflammasome-forming PRRs oligomerize with the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) to generate a large structure in the cytosol, which induces the dimerization, autoproteolysis, and activation of the pro-caspase-1 zymogen. However, several PRRs can also directly interact with pro-caspase-1 without ASC, forming smaller "ASC-independent" inflammasomes. It is currently thought that little, if any, pro-caspase-1 autoproteolysis occurs during, and is not required for, ASC-independent inflammasome signaling. Here, we show that the related human PRRs NLRP1 and CARD8 exclusively form ASC-dependent and ASC-independent inflammasomes, respectively, identifying CARD8 as the first canonical inflammasome-forming PRR that does not form an ASC-containing signaling platform. Despite their different structures, we discovered that both the NLRP1 and CARD8 inflammasomes require pro-caspase-1 autoproteolysis between the small and large catalytic subunits to induce pyroptosis. Thus, pro-caspase-1 self-cleavage is a required regulatory step for pyroptosis induced by human canonical inflammasomes.

DPP8/9 inhibitors are universal activators of functional NLRP1 alleles.

Intracellular pathogenic structures or activities stimulate the formation of inflammasomes, which recruit and activate caspase-1 and trigger an inflammatory form of cell death called pyroptosis. The well-characterized mammalian inflammasome sensor proteins all detect one specific type of signal, for example double-stranded DNA or bacterial flagellin. Remarkably, NLRP1 was the first protein discovered to form an inflammasome, but the pathogenic signal that NLRP1 detects has not yet been identified. NLRP1 is highly polymorphic, even among inbred rodent strains, and it has been suggested that these diverse NLRP1 alleles may have evolved to detect entirely different stimuli. Intriguingly, inhibitors of the serine proteases DPP8 and DPP9 (DPP8/9) were recently shown to activate human NLRP1, its homolog CARD8, and several mouse NLRP1 alleles. Here, we show now that DPP8/9 inhibitors activate all functional rodent NLRP1 alleles, indicating that DPP8/9 inhibition induces a signal detected by all NLRP1 proteins. Moreover, we discovered that the NLRP1 allele sensitivities to DPP8/9 inhibitor-induced and Toxoplasma gondii-induced pyroptosis are strikingly similar, suggesting that DPP8/9 inhibition phenocopies a key activity of T. gondii. Overall, this work indicates that the highly polymorphic NLRP1 inflammasome indeed senses a specific signal like the other mammalian inflammasomes.

N-terminal degradation activates the NLRP1B inflammasome.

Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome.

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.

DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.

6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer.

Metastasis is the cause of death in the majority (∼90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5 -N, N-(hexamethylene)amiloride (HMA) reportedly show robust antitumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases, and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and antikaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model, and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.

General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases.

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.