Biology subjects

Haraldsson, M.

Publications and source records attributed to Haraldsson, M..

7 recordsLinked to original sources

bioRxiv2026

Proteomic profiling reveals pleiotropic antimetabolite activity of triciribine in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) exhibits marked genetic and metabolic heterogeneity that limits the efficacy of targeted therapies. Antimetabolite strategies remain central to ALL treatment, yet the mechanisms underlying differential drug sensitivity are incompletely defined. Here, we investigated the activity of the purine analog triciribine (TCN) across diverse ALL cellular models. We find that TCN exerts potent cytotoxic effects in multiple ALL cell lines, exceeding those observed with canonical Akt inhibitors in our datasets. Phosphoproteomic analyses indicate that, at early time points, TCN does not primarily suppress Akt signaling but instead induces transient pathway activation accompanied by inhibition of cyclin-dependent kinases in both sensitive and resistant cells. The monophosphorylated metabolite TCN-P represents the predominant intracellular species and requires adenosine kinase (ADK) for activity, with ADK protein levels positively correlating with TCN sensitivity in both cell lines and primary patient samples. Using Proteome Integral Solubility Alteration profiling, we identify candidate protein interactions of TCN-P distributed across multiple cellular pathways, including nucleotide metabolism, DNA replication, and protein synthesis. These interactions are accompanied by impaired purine biosynthesis, DNA damage, translational stress, and cell-cycle arrest, as supported by time-course quantitative proteomics and immunoblot analyses. Together, these findings characterize triciribine as an antileukemic agent with pleiotropic antimetabolite activity in ALL and highlight ADK-dependent metabolism as a key determinant of therapeutic sensitivity, suggesting that ADK levels may serve as a predictive biomarker to stratify patients for triciribine-based precision treatment strategies.

cancer biology↗

bioRxiv2026

A chemical inducer of ribophagy limits the toxicity of ALS-related arginine-rich peptides

C9ORF72 intronic repeat expansions are the most frequent mutation found in Amyotrophic Lateral Sclerosis (ALS), producing toxic arginine-rich dipeptides (DPR) that disrupt RNA metabolism and trigger the accumulation of orphan ribosomal proteins (RP). Through a large phenotypic chemical screen, we identified "SALSa", a novel compound that mitigates DPR toxicity. Mechanistically, SALSa acts as a chemical inducer of ribophagy, a specialized form of autophagy that promotes RP clearance. Interestingly, this effect is unrelated to mTOR inhibition, the main regulator of autophagy. In contrast, this is due to an effect of the drug in ribosome biogenesis, which triggers a protective response to clear defective ribosomes. Accordingly, SALSa accumulates in nucleoli and perturbs the final steps of rRNA maturation. SALSa reduces DPR toxicity in differentiated neurons and significantly extends lifespan in a Drosophila melanogaster model of C9ORF72 ALS. These findings suggest that stimulating ribophagy could be beneficial for pathologies associated to dysfunctional ribosome biogenesis, including C9ORF72 ALS.

molecular biology↗

bioRxiv2025

Disrupting Notch signalling by a small molecule inhibiting dihydroorotate dehydrogenase activity

The Notch signalling pathway is highly evolutionarily conserved and regulates differentiation and homeostasis in most organs. Given the critical role of Notch signalling for normal development, dysregulated Notch signalling is frequently linked to pathogenesis of disease and cancer. Hence, developing Notch-targeting therapeutics is warranted but has been challenging and Notch inhibitors have not yet reached broad clinical use. In this report, we identify potential Notch inhibitors, using a novel cell-based Notch reporter system for unbiased screening of compounds reducing Notch signalling. A library of 37.966 small organic compounds was screened for inhibitor candidates, followed by a counter screen to eliminate {gamma}-secretase inhibitor-like compounds and an orthogonal screen based on the role of Notch signalling in myogenic differentiation. This triage led to the identification of five Notch inhibitor candidate hits with different chemical backbones and unrelated to previous Notch antagonists. One candidate hit showed structural similarities to dihydroorotate dehydrogenase (DHODH) inhibitors, and we provide evidence that inhibition of DHODH activity reduces Notch signalling. In conclusion, our data support the notion that DHODH inhibition may be an interesting avenue to explore for the development of novel Notch inhibitors.

cancer biology↗

bioRxiv2024

ABHD2 activity is not required for the non-genomic action of progesterone on human sperm

Study questionIs the hydrolase ABHD2 required for progesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm? Summary answerProgesterone-induced Ca2+ influx via CatSper and the resulting motility responses in human sperm do not require ABHD2 activity. What is known alreadySperm motility is tightly regulated by signalling pathways that are activated as sperm ascend the female reproductive tract, including progesterone triggering Ca2+ influx via the CatSper channel and inducing hyperactivated motility needed for fertilization. This process is thought to involve ABHD2, which may hydrolyse the endogenous CatSper inhibitor 2-arachidonoylglycerol (2-AG), thereby relieving inhibition and enabling calcium entry into the flagellum. Study design, size, durationPotent small molecule inhibitors of ABHD2 activity were synthesized, characterized, and used as tools to scrutinize the role of ABHD2 in activation of CatSper and regulation of sperm motility. Participants/materials, setting, methodsDerivatives of published ABHD2 inhibitors were optimised for in vitro potency and cellular activity and subsequently tested in human sperm motility and Ca2+ influx assays. Main results and the role of chanceProgesterone does not activate ABHD2 in vitro. In addition, inhibition of ABHD2 in human sperm has no effect on progesterone-induced Ca2+ influx through CatSper nor on basal or progesterone-induced hyperactivated motility. This demonstrates that ABHD2 activity is, in fact, not required for the non-genomic action of progesterone on human sperm. Large scale datanone Limitations, reasons for cautionWe examined the effects of inhibition of the enzymatic activity of ABHD2. We cannot exclude that ABHD2 functions as a part of a larger multiprotein complex, in which it may play a structural role independent of its hydrolase activity. Wider implications of the findingsThis study presents conclusive evidence that ABHD2 does not bind progesterone and that its hydrolase activity is not required for progesterone activation of CatSper and resulting changes in motility of human sperm. These results highlight the need for further research to elucidate the mechanism underlying the non-genomic action of progesterone on human sperm. Study funding/competing interest(s)This publication is based on research funded by the Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Gates Foundation. LT, CB, and TS were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - project numbers 329621271 (CRU326; CB, TS), and 404595355 (Research Training Group "Chemical biology of ion channels (Chembion); LT, TS). This work was funded by the German Federal Ministry of Education and Research (BMBF) within the framework of Contraception Research, grant number 01GR2501A and 01GR2502A. A.A., K.V., A.T., N.D., A.H., M.W. and R.L. are employees of Nuvisan ICB GMBH, Berlin, Germany. Nuvisan is recipient of a Gates Foundation grant. A.A is associate editor of Human Reproduction Open.

cell biology↗

bioRxiv2023

Discovery of a novel inhibitor of macropinocytosis with antiviral activity

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a new molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using High-Throughput Microscopy, where we identified new chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as Monkeypox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present a new molecule that inhibits viral entry via the endocytic route, offering a new alternative to prevent viral infection.

pharmacology and toxicology↗

bioRxiv2023

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1 via a comprehensive screening funnel

Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase central to cellular nucleotide pool homeostasis. Recent literature has also demonstrated how SAMHD1 can detoxify chemotherapy metabolites thereby controlling their clinical responses. To further understand SAMHD1 biology and to investigate the potential of targeting this enzyme as a neoadjuvant to existing chemotherapies we set out to discover selective small molecule-based inhibitors of SAMHD1. Here we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for further characterisation of the screen output. The identified hit compound TH6342 and its analogues, accompanied by their inactive negative control analogue TH7126, demonstrated specific, low M potency in inhibiting the hydrolysis of both natural substrates and nucleotide analogue therapeutics, shown using complementary enzyme-coupled and direct enzymatic activity assays. Their mode of inhibition was subsequently detailed by coupling kinetic studies with thermal shift assays, where TH6342 and analogues were shown to engage with pre-tetrameric SAMHD1 and deter the oligomerisation and allosteric activation of SAMHD1 without occupying nucleotide binding pockets. We further outline the development and application of multiple cellular assays for assessing cellular target engagement and associated functional effects, including CETSA and an in-cell dNTP hydrolase activity assay, which highlighted future optimisation strategies of this chemotype. In summary, with a novel mode of inhibition, TH6342 and analogues broaden the set of tool compounds available in deciphering SAMHD1 enzymology and functions, and furthermore, the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/524275v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc706forg.highwire.dtl.DTLVardef@59a9b4org.highwire.dtl.DTLVardef@9473e2org.highwire.dtl.DTLVardef@4427dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

cell biology↗