A quantitative high-throughput screen identifies compounds that lower expression of the SCA2-and ALS-associated gene ATXN2.

CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS.

Convergent olfactory trace amine-associated receptors detect biogenic polyamines with distinct motifs via a conserved binding site.

Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp3.32 and Tyr6.51 residues. sTAAR365, which has remarkable sensitivity for cadaverine (EC50 = 4 nM), uses an extra residue, Thr7.42, to stabilize ligand binding. These cadaverine recognition sites also interact with amines with four and three amino groups (spermine and spermidine, respectively). Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling.

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy.

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson's disease pathology.

Spermine in semen of male sea lamprey acts as a sex pheromone.

Semen is fundamental for sexual reproduction. The non-sperm part of ejaculated semen, or seminal plasma, facilitates the delivery of sperm to the eggs. The seminal plasma of some species with internal fertilization contains anti-aphrodisiac molecules that deter promiscuity in post-copulatory females, conferring fitness benefits to the ejaculating male. By contrast, in some taxa with external fertilization such as fish, exposure to semen promotes spawning behaviors. However, no specific compounds in semen have been identified as aphrodisiac pheromones. We sought to identify a pheromone from the milt (fish semen) of sea lamprey (Petromyzon marinus), a jawless fish that spawns in lek-like aggregations in which each spermiating male defends a nest, and ovulatory females move from nest to nest to mate. We postulated that milt compounds signal to ovulatory females the presence of spawning spermiating males. We determined that spermine, an odorous polyamine initially identified from human semen, is indeed a milt pheromone. At concentrations as low as 10-14 molar, spermine stimulated the lamprey olfactory system and attracted ovulatory females but did not attract males or pre-ovulatory females. We found spermine activated a trace amine-associated receptor (TAAR)-like receptor in the lamprey olfactory epithelium. A novel antagonist to that receptor nullified the attraction of ovulatory females to spermine. Our results elucidate a mechanism whereby a seminal plasma pheromone attracts ready-to-mate females and implicates a possible conservation of the olfactory detection of semen from jawless vertebrates to humans. Milt pheromones may also have management implications for sea lamprey populations.

Two highly related odorant receptors specifically detect α-bile acid pheromones in sea lamprey (Petromyzon marinus).

Pheromones play critical roles in habitat identification and reproductive behavior synchronization in the sea lamprey (Petromyzon marinus). The bile acid 3-keto petromyzonol sulfate (3kPZS) is a major component of the sex pheromone mixture from male sea lamprey that induces specific olfactory and behavioral responses in conspecific individuals. Olfactory receptors interact directly with pheromones, which is the first step in their detection, but identifying the cognate receptors of specific pheromones is often challenging. Here, we deorphanized two highly related odorant receptors (ORs), OR320a and OR320b, of P. marinus that respond to 3kPZS. In a heterologous expression system coupled to a cAMP-responsive CRE-luciferase, OR320a and OR320b specifically responded to C24 5α-bile acids, and both receptors were activated by the same set of 3kPZS analogs. OR320a displayed larger responses to all 3kPZS analogs than did OR320b. This difference appeared to be largely determined by a single amino acid residue, Cys-792.56, the C-terminal sixth residue relative to the most conserved residue in the second transmembrane domain (2.56) of OR320a. This region of TM2 residues 2.56-2.60 apparently is critical for the detection of steroid compounds by odorant receptors in lamprey, zebrafish, and humans. Finally, we identified OR320 orthologs in Japanese lamprey (Lethenteron camtschaticum), suggesting that the OR320 family may be widely present in lamprey species and that OR320 may be under purifying selection. Our results provide a system to examine the origin of olfactory steroid detection in vertebrates and to define a highly conserved molecular mechanism for steroid-ligand detection by G protein-coupled receptors.

Neuroprotection and repair of 3'-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1).

Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3' ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5' UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3'-DNA-tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses.

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

Combination screen in multi-cell type tumor spheroids reveals interaction between aryl hydrocarbon receptor antagonists and E1 ubiquitin-activating enzyme inhibitor: Aryl-hydrocarbon receptor antagonist drug combinations.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.

HTS384 NCI60: The Next Phase of the NCI60 Screen.

The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for more than 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. In this study, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. Although the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1,003 FDA-approved and investigational small-molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the NCI to facilitate drug discovery by the cancer research community. Significance: The new NCI60 cell line screen HTS384 shows robust patterns of response to oncology agents and substantial overlap with the classic screen, providing an updated tool for studying therapeutic agents. See related commentary by Colombo and Corsello, p. 2397.