ToxProfiler: A novel human-based reporter assay for in vitro chemical safety assessment.

In vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurements and fails to adequately predict human toxicity. To improve the in vitro chemical safety assessment, it is important to understand the underlying mechanisms of toxicity. Here we introduce ToxProfiler, a novel human-based reporter assay that quantifies the chemical-induced stress responses at a single-cell level and reveals the toxicological mode-of-action (MoA) of novel drugs and chemicals. The assay accurately measures the activation of seven major cellular stress response pathways (oxidative stress, cell cycle stress, endoplasmic reticulum stress, ion stress, protein stress, autophagy and inflammation) that play a role in the adaptive responses prior to cellular toxicity. To assess the applicability of the assay in predicting the toxicity MoA of chemicals, we tested a set of 100 chemicals with well-known in vitro and in vivo toxicological profiles. Concentration response modeling and point-of-departure estimation for each reporter protein allowed for chemical potency ranking and revealed the primary toxicological MoA of chemicals. Furthermore, the assay could effectively group chemicals based on their shared toxicity signatures and link them to specific toxicological targets, e.g. mitochondrial toxicity and genotoxicity, and different human pathologies, including liver toxicity and cardiotoxicity. Overall, ToxProfiler is a quantitative in vitro reporter assay that can accurately provide insight into the toxicological MoA of compounds, thereby assisting in the future mechanism-based safety assessment of chemicals.

Quantitative interpretation of ToxTracker dose-response data for potency comparisons and mode-of-action determination.

ToxTracker is an in vitro mammalian stem cell-based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose-response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold-change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined-covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose-response modeling of multivariate ToxTracker data can be used for potency ranking and mode-of-action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment.

Discovery of the Potent and Selective Inhaled Janus Kinase 1 Inhibitor AZD4604 and Its Preclinical Characterization.

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata.

Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.

TubulinTracker, a Novel In Vitro Reporter Assay to Study Intracellular Microtubule Dynamics, Cell Cycle Progression, and Aneugenicity.

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.

Genotoxicity assessment of potentially mutagenic nucleoside analogues using ToxTracker®.

Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues.

A novel human stem cell-based biomarker assay for in vitro assessment of developmental toxicity.

BACKGROUND: Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time-consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity. METHODS: To identify embryotoxic compounds, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay. The assay is based on the differentiation of hiPSCs into functional cardiomyocytes and hepatocytes. Proper stem cell differentiation is investigated by morphological profiling and assessment of time-dependent expression patterns of cell-specific biomarkers. In this system, a decrease in the expression of the biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity. RESULTS: The hiPSCs-based biomarker assay was validated with 21 well-established in vivo animal teratogenic and non-teratogenic compounds during cardiomyocyte and hepatocyte differentiation. The in vivo teratogenic compounds (e.g., thalidomide and valproic acid) markedly disrupted morphology, functionality, and the expression pattern of the biomarker genes in either one or both cell types. Non-teratogenic chemicals generally had no effect on the morphology of differentiated cells, nor on the expression of the biomarker genes. Compared to the in vivo classification, the assay achieved high accuracy (91%), sensitivity (91%), and specificity (90%). CONCLUSION: The assay, which we named ReproTracker®, is a state-of-the-art in vitro method that can identify the teratogenicity potential of new pharmaceuticals and chemicals and signify the outcome of in vivo test systems.

Dual function of UNC-51-like kinase 3 (Ulk3) in the Sonic hedgehog signaling pathway.

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.

Hedgehog signalling: how to get from Smo to Ci and Gli.

The secreted morphogens of the Hedgehog family have important roles in normal development as well as in associated pathologies, including cancer. The Hedgehog signalling pathway has been studied in Drosophila and is thought to be conserved in vertebrates. Hedgehog elicits a signalling response that activates Smoothened (Smo). There is evidence of differences between Drosophila and vertebrates concerning signalling downstream of Smo, as well as in Smo itself. Here, we discuss this evidence and its importance for investigations of the pathway and related biology, as well as for the development of drugs targeting components of the pathway for treatment of associated pathologies.

Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway.

The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.

Melanocortin 1 Receptor Agonists Based on a Bivalent, Bicyclic Peptide Framework.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In in vitro binding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50 > 10.3; EC50 < 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1.

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p Ki of 8.73 ± 0.08 ( Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

The FU gene and its possible protein isoforms.

BACKGROUND: FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. RESULTS: The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. CONCLUSIONS: The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.

The transcriptional repressor domain of Gli3 is intrinsically disordered.

The transcription factor Gli3 is acting mainly as a transcriptional repressor in the Sonic hedgehog signal transduction pathway. Gli3 contains a repressor domain in its N-terminus from residue G106 to E236. In this study we have characterized the intracellular structure of the Gli3 repressor domain using a combined bioinformatics and experimental approach. According to our findings the Gli3 repressor domain while being intrinsically disordered contains predicted anchor sites for partner interactions. The obvious interaction partners to test were Ski and DNA; however, with both of these the structure of Gli3 repressor domain remained disordered. To locate residues important for the repressor function we mutated several residues within the Gli3 repressor domain. Two of these, H141A and H157N, targeting predicted helical regions, significantly decreased transcriptional repression and thus identify important functional parts of the domain.

Identification of the gene transcription repressor domain of Gli3.

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

A possible role of mouse Fused (STK36) in Hedgehog signaling and Gli transcription factor regulation.

The segment polarity gene Fused (Fu) encodes a putative serine-threonine kinase Fu, which has been shown to play a key role in the Hedgehog signaling pathway of Drosophila. Human FU (hFU) has been shown to enhance the activity of Gli transcription factors, targets of the signaling pathway. However, Fu ( -/- ) mice do not show aberrant embryonic development indicating that mouse Fu (mFu) is dispensable for Hedgehog signaling until birth. In order to investigate if there are important differences between hFU and mFu, we cloned the cDNA, analyzed expression and tested the ability of mFu to regulate Gli proteins. Of the tested tissues only brain and testis showed significant expression. However, in transient overexpression analyses mFu was able to enhance Gli induced transcription in a manner similar to hFU. Thus, we turned to RNAi in order to test if mFu would be important for Hedgehog signaling after all. In one cell line with reduced mFu expression the Hedgehog signaling was severely hampered, indicating that mFu may have a role in Hedgehog signaling and Gli regulation in some cellular situations.