Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease.

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

Apoptosis assessment in high-content and high-throughput screening assays.

Here the authors describe the development of AUTOptosis, an economical and rapid apoptosis monitoring method suitable for high-content and high-throughput screening assays. AUTOptosis is based on the quantification of nuclei intensity via staining with Hoechst 33342. First, the authors calibrated the method using standard apoptosis inducers in multiple cell lines. Next, the authors validated the applicability of this approach to high-content screening using a small library of compounds and compared it with the terminal deoxynucleotidyl transferase dUTP nick end labeling gold standard. Finally, the authors demonstrated the specificity of the method by using AUTOposis to detect apoptosis triggered by Mycobacterium tuberculosis intracellular infections.

High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis.

A screen of a eukaryotic kinase inhibitor library in an established intracellular infection model identified a set of drug candidates enabling intracellular killing of Mycobacterium tuberculosis (M.tb). Screen validity was confirmed internally by a Z' = 0.5 and externally by detecting previously reported host-targeting anti-M.tb compounds. Inhibitors of the CHK kinase family, specifically checkpoint kinase 2 (CHK2), showed the highest inhibition and lowest toxicity of all kinase families. The screen identified and validated DDUG, a CHK2 inhibitor, as a novel bactericidal anti-M.tb compound. CHK2 inhibition by RNAi phenocopied the intracellular inhibitory effect of DDUG. DDUG was active intracellularly against M.tb, but not other mycobacteria. DDUG also had extracellular activity against 4 of 12 bacteria tested, including M.tb. Combined, these observations suggest DDUG acts in tandem against both host and pathogen. Importantly, DDUG's validation highlights the screening and analysis methodology developed for this screen, which identified novel host-directed anti-M.tb compounds.

Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening.

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency.

Highly active antiretroviral therapy (HAART) has improved the outlook for the HIV epidemic, but does not provide a cure. The proposed "shock-and-kill" strategy is directed at inducing latent HIV reservoirs, which may then be purged via boosted immune response or targeting infected cells. We describe five novel compounds that are capable of reversing HIV latency without affecting the general T-cell activation state. The new compounds exhibit synergy for reactivation of latent provirus with other latency-reversing agents (LRAs), in particular ingenol-3-angelate/PEP005. One compound, designated PH02, was efficient at reactivating viral transcription in several cell lines bearing reporter HIV-1 at different integration sites. Furthermore, it was capable of reversing latency in resting CD4+ T lymphocytes from latently infected aviremic patient cells on HAART, while producing minimal cellular toxicity. The combination of PH02 and PEP005 produces a strong synergistic effect for reactivation, as demonstrated through a quantitative viral outgrowth assay (qVOA), on CD4+ T lymphocytes from HIV-1-infected individuals. We propose that the PH02/PEP005 combination may represent an effective novel treatment for abrogating persistent HIV-1 infection.

Achyrodimer F, a tyrosyl-DNA phosphodiesterase I inhibitor from an Australian fungus of the family Cortinariaceae.

Mass-guided isolation of the dichloromethane/methanol extracts from a specimen of teleomorphic fungus of the family Cortinariaceae resulted in the identification of a new dimeric cyclobutane metabolite, achyrodimer F (1), along with the monomers hispidin (2) and bisnoryangonin (3). Their structures were determined by NMR and MS data analyses. Density Function Theory (DFT) NMR calculations was employed to confirm the chemical structure of achyrodimer F. Compound 1 inhibited tyrosyl-DNA phosphodiesterase I with an IC50 value of 1µM.

Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes.

The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer's Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 µM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRß, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 µM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists.

Novel small molecules potentiate premature termination codon readthrough by aminoglycosides.

Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.

Novel spirothiazamenthane inhibitors of the influenza A M2 proton channel.

The development of treatments for influenza that inhibit the M2 proton channel without being susceptible to the widespread resistance mechanisms associated with the adamantanes is an ongoing challenge. Using a yeast high-throughput yeast growth restoration assay designed to identify M2 channel inhibitors, a single screening hit was uncovered. This compound (3), whose structure was incorrectly identified in the literature, is an inhibitor with similar potency to amantadine against WT M2. A library of derivatives of 3 was prepared and activity against WT M2 and the two principal mutant strains (V27A and S31N) was assessed in the yeast assay. The best compounds were further evaluated in an antiviral plaque reduction assay using engineered WT, V27A and S31N M2 influenza A strains with otherwise identical genetic background. Compound 63 was found to inhibit all three virus strains in this cell based antiviral assay at micromolar concentrations, possibly through a mechanism other than M2 inhibition.

Tyrosyl-DNA Phosphodiesterase I Inhibitors from the Australian Plant Macropteranthes leichhardtii.

Mass-directed isolation of the CH2Cl2/MeOH extract from the bark of an Australian plant, Macropteranthes leichhardtii, resulted in the purification of a new phenylpropanoid glucoside, macropteranthol (1), together with four known analogues (2-5). The structure of compound 1 was elucidated by NMR and MS data analyses and quantum chemical calculations. Compounds 3 and 5 showed inhibitory activity against tyrosyl-DNA phosphodiesterase I with IC50 values of ∼1.0 µM.

Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B.

An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B. Substrate cleavage was readily monitored and was linear for up to 4h but enzyme concentrations of about ten-fold higher were required compared to assays using protein substrate LC3 or analogs thereof (such as FRET-LC3). Several known inhibitors of ATG4B were evaluated using the small peptide substrate and gave IC50 values 3-7 fold higher than previously obtained values using the FRET-LC3 substrate.

Small organic molecule disruptors of Cav3.2 - USP5 interactions reverse inflammatory and neuropathic pain.

BACKGROUND: Cav3.2 channels facilitate nociceptive transmission and are upregulated in DRG neurons in response to nerve injury or peripheral inflammation. We reported that this enhancement of Cav3.2 currents in afferent neurons is mediated by deubiquitination of the channels by the deubiquitinase USP5, and that disrupting USP5/Cav3.2 channel interactions protected from inflammatory and neuropathic pain. RESULTS: Here we describe the development of a small molecule screening assay for USP5-Cav3.2 disruptors, and report on two hits of a ~5000 compound screen - suramin and the flavonoid gossypetin. In mouse models of inflammatory pain and neuropathic pain, both suramin and gossypetin produced dose-dependent and long-lasting mechanical anti-hyperalgesia that was abolished or greatly attenuated in Cav3.2 null mice. Suramin and Cav3.2/USP5 Tat-disruptor peptides were also tested in models of diabetic neuropathy and visceral pain, and provided remarkable protection. CONCLUSIONS: Overall, our findings provide proof of concept for a new class of analgesics that target T-type channel deubiquitination.

Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.

Identification of a putative Tdp1 inhibitor (CD00509) by in vitro and cell-based assays.

Mutations of DNA repair pathways contribute to tumorigenesis and provide a therapeutic target for synthetic lethal interactions in tumor cells. Given that tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs stalled topoisomerase-I DNA complexes, we hypothesized that inhibition of Tdp1 has synthetic lethal effects in some cancers. To test this, we screened tumor arrays for Tdp1 expression and observed that Tdp1 is expressed in many tumors, including more than 90% of human breast tumors. Subsequent chemical screening identified putative Tdp1 inhibitors. Treatment of control human mammary epithelial cells and the breast cancer cell line MCF-7 with compound CD00509 preferentially sensitized MCF-7 cells to camptothecin and decreased cell proliferation 25% more than camptothecin treatment alone. This suggests that CD00509 specifically targeted Tdp1 in vitro, and CD00509 increased the sensitivity of wild-type murine embryonic fibroblasts (MEFs) to camptothecin to a degree comparable to that of Tdp1(-/-) MEFs. In addition, consistent with poly ADP-ribose polymerase-1 (PARP-1) collaborating with Tdp1 in DNA repair, combined Tdp1 and PARP-1 inhibition was more detrimental to MCF-7 cells than either treatment alone, whereas the combination was not additively harmful to control mammary cells. We conclude that targeting Tdp1 in anticancer therapy preferentially enhances the sensitivity of some breast cancer cells to camptothecin and may be an effective adjuvant for breast cancer therapy.

ApoE secretion modulating bromotyrosine derivative from the Australian marine sponge Callyspongia sp.

High throughput screening of a pre-fractionated natural product library identified 11 active fractions showing ApoE modulation activity. Mass-directed fractionation of one active crude extract from the Australian marine sponge Callyspongia sp. resulted in the isolation of 13 metabolites, including three new bromotyrosine derivatives, callyspongic acid (1), 3,5-dibromo-4-methoxyphenylpyruvic acid (2), N-acetyl-3-bromo-4-hydroxylphenylethamine (3), and ten known compounds (4-13). The structure elucidation of compounds 1-3 was based on their 1D and 2D NMR and MS spectroscopic data. 3,5-Dibromo-4-methoxyphenylpyruvic acid (2) showed weak activity in increasing the apolipoprotein E secretion from human CCF-STTG1 cells at the concentration of 40 µM.

Development of fluorescent substrates and assays for the key autophagy-related cysteine protease enzyme, ATG4B.

The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.

Aplysinellamides A-C, bromotyrosine-derived metabolites from an Australian Aplysinella sp. marine sponge.

Mass-directed fractionation of an extract from the Australian marine sponge Aplysinella sp., from the Great Barrier Reef, resulted in the isolation of four new bromotyrosine derivatives, aplysinellamides A-C (1-3) and aplysamine-1-N-oxide (4), along with six known compounds (5-10). The structure elucidation of compounds 1-4 was based on their 1D and 2D NMR and MS spectroscopic data. Aplysamine-1 (6) increased the apolipoprotein E secretion from human CCF-STTG1 astrocytoma cells by 2-fold at the concentration of 30 µM.

Hormonal modulators of glial ABCA1 and apoE levels.

Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.

An evolutionarily conserved synthetic lethal interaction network identifies FEN1 as a broad-spectrum target for anticancer therapeutic development.

Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.