Polycystin-1 Interacting Protein-1 (CU062) Interacts with the Ectodomain of Polycystin-1 (PC1).

The PKD1 gene, encoding protein polycystin-1 (PC1), is responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 has been shown to be present in urinary exosome-like vesicles (PKD-ELVs) and lowered in individuals with germline PKD1 mutations. A label-free mass spectrometry comparison of urinary PKD-ELVs from normal individuals and those with PKD1 mutations showed that several proteins were reduced to a degree that matched the decrease observed in PC1 levels. Some of these proteins, such as polycystin-2 (PC2), may be present in a higher-order multi-protein assembly with PC1-the polycystin complex (PCC). CU062 (Q9NYP8) is decreased in ADPKD PKD-ELVs and, thus, is a candidate PCC component. CU062 is a small glycoprotein with a signal peptide but no transmembrane domain and can oligomerize with itself and interact with PC1. We investigated the localization of CU062 together with PC1 and PC2 using immunofluorescence (IF). In nonconfluent cells, all three proteins were localized in close proximity to focal adhesions (FAs), retraction fibers (RFs), and RF-associated extracellular vesicles (migrasomes). In confluent cells, primary cilia had PC1/PC2/CU062 + extracellular vesicles adherent to their plasma membrane. In cells exposed to mitochondrion-decoupling agents, we detected the development of novel PC1/CU062 + ring-like structures that entrained swollen mitochondria. In contact-inhibited cells under mitochondrial stress, PC1, PC2, and CU062 were observed on large, apically budding extracellular vesicles, where the proteins formed a reticular network on the membrane. CU062 interacts with PC1 and may have a role in the identification of senescent mitochondria and their extrusion in extracellular vesicles.

Analysis of the polycystin complex (PCC) in human urinary exosome-like vesicles (ELVs).

The polycystin-1 (PC1), polycystin-2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome-like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C-terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C-terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C-terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is  > 2 MDa in size and that N-terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.

Characterization of Lead Compounds Targeting the Selenoprotein Thioredoxin Glutathione Reductase for Treatment of Schistosomiasis.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1-5 are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1-5, 7, and 8 displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds 1-4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds 4-6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

Human-Specific Abnormal Alternative Splicing of Wild-Type PKD1 Induces Premature Termination of Polycystin-1.

BACKGROUND: The major form of autosomal dominant polycystic kidney disease is caused by heterozygous mutations in PKD1, the gene that encodes polycystin-1 (PC1). Unlike PKD1 genes in the mouse and most other mammals, human PKD1 is unusual in that it contains two long polypyrimidine tracts in introns 21 and 22 (2.5 kbp and 602 bp, respectively; 97% cytosine and thymine). Although these polypyrimidine tracts have been shown to form thermodynamically stable segments of triplex DNA that can cause DNA polymerase stalling and enhance the local mutation rate, the efficiency of transcription and splicing across these cytosine- and thymine-rich introns has been unexplored. METHODS: We used RT-PCR and Western blotting (using an mAb to the N terminus) to probe splicing events over exons 20-24 in the mouse and human PKD1 genes as well as Nanopore sequencing to confirm the presence of multiple splice forms. RESULTS: Analysis of PC1 indicates that humans, but not mice, have a smaller than expected protein product, which we call Trunc_PC1. The findings show that Trunc_PC1 is the protein product of abnormal differential splicing across introns 21 and 22 and that 28.8%-61.5% of PKD1 transcripts terminate early. CONCLUSIONS: The presence of polypyrimidine tracts decreases levels of full-length PKD1 mRNA from normal alleles. In heterozygous individuals, low levels of full-length PC1 may reduce polycystin signaling below a critical "cystogenic" threshold.

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

A new epitope-tagged Pkhd1 allele sheds light on fibrocystin signaling.

In this issue of Kidney International, Outeda et al. present a new epitope-tagged allele of murine Pkhd1 that allows the monitoring of functional fibrocystin in vivo from the extreme C-terminus of the molecule. This work also shows that the removal of two-thirds of the intracellular tail of fibrocystin does not result in cystogenesis in either the liver or kidney, with major implications for our understanding of Pkhd1 function and polycystic kidney disease in general.

Correction: A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

[This corrects the article DOI: 10.1371/journal.pone.0161486.].

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins.

Discovery of a novel general anesthetic chemotype using high-throughput screening.

BACKGROUND: The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. METHODS: Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. RESULTS: From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. CONCLUSION: The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Biochemical assays for the discovery of TDP1 inhibitors.

Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 µm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 µm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

Peroxiredoxin-1 from the human hookworm Ancylostoma ceylanicum forms a stable oxidized decamer and is covalently inhibited by conoidin A.

Hookworms are parasitic nematodes that have a devastating impact on global health, particularly in developing countries. We report a biochemical and structural analysis of a peroxiredoxin from the hookworm Ancylostoma ceylanicum, AcePrx-1. Peroxiredoxins provide antioxidant protection and act as signaling molecules and chaperones. AcePrx-1 is expressed in adult hookworms and can be inactivated by 2,3-bis(bromomethyl)quinoxaline-1,4-dioxide (conoidin A). Conoidin A inactivates AcePrx-1 by alkylating or crosslinking the catalytic cysteines, while maintaining the enzyme in the "locally unfolded" conformation. Irreversible oxidation of the resolving cysteine may contribute additional inhibitory activity. A crystal structure of oxidized AcePrx-1 reveals a disulfide-linked decamer. A helix macrodipole near the active site increases the reactivity of the catalytic cysteines to conoidin A. This work demonstrates the promise of conoidin compounds as probes to evaluate peroxiredoxins as drug targets in human parasites.

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

High-throughput screen identifies cyclic nucleotide analogs that inhibit prostatic acid phosphatase.

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5'-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well-based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC(1280)) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter.

Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays, owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes, including FLuc-catalyzed formation of multisubstrate adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays, compounds acting as FLuc inhibitors yield paradoxical luminescence increases, thus data on compounds acquired from FLuc-dependent assays require careful analysis as described here.

Discovery of a novel noniminosugar acid α glucosidase chaperone series.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA). Many disease-causing mutated GAA retain enzymatic activity but are not translocated from endoplasmic reticulum (ER) to lysosomes. Enzyme replacement therapy (ERT) is the only treatment for Pompe disease but remains expensive, inconvenient, and does not reverse all disease manifestations. It was postulated that small molecules which aid in protein folding and translocation to lysosomes could provide an alternate to ERT. Previously, several iminosugars have been proposed as small-molecule chaperones for specific LSDs. Here we identified a novel series of noniminosugar chaperones for GAA. These moderate GAA inhibitors are shown to bind and thermostabilize GAA and increase GAA translocation to lysosomes in both wild-type and Pompe fibroblasts. AMDE and physical properties studies indicate that this series is a promising lead for further pharmacokinetic evaluation and testing in Pompe disease models.

Discovery, structure-activity relationship, and biological evaluation of noninhibitory small molecule chaperones of glucocerebrosidase.

A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.

Non-iminosugar glucocerebrosidase small molecule chaperones.

Small molecule chaperones are a promising therapeutic approach for the Lysosomal Storage Disorders (LSDs). Here, we report the discovery of a new series of non-iminosugar glucocerebrosidase inhibitors with chaperone capacity, and describe their structure activity relationship (SAR), selectivity, cell activity phamacokinetics.