Protein prenylation is a post-translational modification promoting membrane association where isoprenoid lipids attach to C-terminal cysteines of eukaryotic proteins such as Ras and Rho GTPases, nucleus lamins, and G-protein subunits. Three enzymes catalyze this process: farnesyltransferase (FTase) and geranylgeranyltransferase type I and II (GGTase I and RabGGTase). FTase and GGTase-I recognize C-terminal CaaX motifs, of which the terminal amino acid confers specificity. Due to its involvement in oncogenic Ras activation, FTase has become a major anticancer target for drug development. Although first-generation FTase inhibitors failed in clinical trials in many cancers due to compensatory geranylgeranylation of KRAS and NRAS, they remain effective against HRAS-driven tumors and other pathologies, such as Hutchinson-Gilford progeria syndrome. The FTase inhibitor A-176120 was reported to compete with farnesyl and not KRAS. However, our crystallographic and biochemical analyses reveal that A-176120 sterically interferes with the engagement of the KRAS CAAX motif, reducing, but not abolishing, its binding to FTase.

The Sec pathway is an essential protein secretion route for all organisms. In bacteria, the SecA ATPase peripherally associates with the SecYEG channel to form the translocase that mediates preprotein export. Activation of the translocase depends strictly on the synergy of signal peptide and mature domain binding. Thus, client selectivity, translocase activation and protein secretion are coupled by one mechanism. We show here that a previously identified small molecule (HSI#6) binds SecA, modulates its intrinsic dynamics and allosterically activates the translocase in the absence of clients. By uncoupling translocase activation from preprotein binding, HSI#6 transformed the translocase into a promiscuous nanomachine that lost client selectivity and secreted unfolded pre- mature- and cytoplasmic- proteins with high efficiency in vivo or in vitro. To our knowledge, HSI#6 is the first activator of the Sec pathway and might offer unique opportunities for the discovery of new antibacterials.

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1-S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We show stereoselectivity of TRPM3 for the (R)-enantiomer of the flavonoid antagonist isosakuranetin and the (R)-enantiomer of the synthetic agonist CIM0216. Mutations within this pocket-including variants identified in patients -alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy.

Parkinson’s disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) are progressive neurodegenerative disorders marked by the pathological aggregation of alpha-synuclein (ɑSyn). Despite significant research efforts, effective therapeutic interventions remain elusive due to limited understanding of the cellular effects of ɑSyn aggregation and propagation. This study presents the development of a scalable cellular seeding assay for screening small molecules targeting cellular ɑSyn seeded aggregation. By leveraging a fluorescent reporter of ɑSyn and phenotypic screening, the assay enables high-throughput evaluation of potential inhibitors in a cellular environment mimicking disease pathology. We evaluated three different αSyn aggregation inhibitors tested in clinical trials for PD: Minzasolmin, Emrusolmin and EGCG and profiled gene expression using multiplexed single cell RNA sequencing in order to examine their distinct effects on cellular pathways associated with ɑSyn overexpression or seeded aggregation. Two cellular activities were prominently affected: lipid metabolism and rRNA processing. Notably, while EGCG effects were confined to cells with aggregated αSyn, Minzasolmin and Emrusolmin also produced transcriptional changes in cells without aggregated αSyn. Each of the compounds tested induced a partial reversal of transcriptional effects resulting from αSyn seeded aggregation. We identified 391 genes that were no longer significantly differentially expressed upon addition of compound, relative to cells with seeded aggregation. This platform bridges phenotypic screening and molecular pathway analysis, providing insights into druggable pathways for synucleinopathies. The molecular signatures identified here can assist in testing and benchmarking future drug discovery leads.

The paramyxovirus family includes important pathogens such as measles and mumps viruses, as well as emerging pathogens with pandemic potential such as Nipah virus. Despite the threat to public health and the frequent identification of novel paramyxoviruses, no antiviral drugs are currently available. A hallmark of most paramyxoviruses is the induction of cell-cell fusion leading to syncytia formation. To facilitate antiviral drug discovery, we leveraged this trait and established a high-throughput split-green fluorescent protein (GFP) antiviral screening assay suitable for high-content imaging through the quantification of virus-induced GFP+ syncytia. The assay was validated with well-known broad-spectrum antiviral compounds against representative members of five different Paramyxovirinae genera. Using this split-GFP assay, a small-molecule repurposing library of approximately 3000 compounds was screened against recombinant Cedar virus (CedV), a nonpathogenic henipavirus. Two molecules were identified: Cathepsin Inhibitor 1 with henipavirus-specific activity and PF-543 with pan-paramyxovirus activity. Both molecules inhibit viral replication by blocking cell-cell fusion. The split-GFP assay presented here will enable the development of extensive drug discovery initiatives aimed at identifying much-needed pan-henipavirus/paramyxovirus inhibitors.

The SARS-CoV-2 outbreak of 2019 had a devastating impact on global health and economies worldwide. The viral cysteine protease (3CLpro) is responsible for viral polypeptide bond cleavages and is therefore an essential target to inhibit viral replication. Here, we report the discovery of an orally available, reversible covalent inhibitor of the SARS-CoV-2 main protease that is also highly active across other human coronaviruses and demonstrated oral efficacy in a Syrian hamster infection model at low plasma concentrations. Projection of pharmacokinetics (PK) in humans, based on PK studies in preclinical species and enhanced in vitro/in vivo efficacy of ALG-097558 (7) indicated the potential for BID dosing without the need for ritonavir, the PK boosting component of Paxlovid. After preclinical safety and pharmacological studies, ALG-097558 has progressed to phase 1 clinical trials.

The coronavirus membrane protein (M) is the main organizer of coronavirus assembly1-3. Here, we report on an M-targeting molecule, CIM-834, that blocks the assembly of SARS-CoV-2. CIM-834 was obtained through high-throughput phenotypic antiviral screening followed by medicinal-chemistry efforts and target elucidation. CIM-834 inhibits the replication of SARS-CoV-2 (including a broad panel of variants) and SARS-CoV. In SCID mice and Syrian hamsters intranasally infected with SARS-CoV-2, oral treatment reduced lung viral titres to nearly undetectable levels, even (as shown in mice) when treatment was delayed until 24 h before the end point. Treatment of infected hamsters prevented transmission to untreated sentinels. Transmission electron microscopy studies show that virion assembly is completely absent in cells treated with CIM-834. Single-particle cryo-electron microscopy reveals that CIM-834 binds and stabilizes the M protein in its short form, thereby preventing the conformational switch to the long form, which is required for successful particle assembly. In conclusion, we have discovered a new druggable target in the replication cycle of coronaviruses and a small molecule that potently inhibits it.

Figure S1 shows A) Fraction of transfected cells after re-expression of NF2 in HEK293 and NF2-mutant mesothelioma cell lines. The proliferation of NF2-mutant mesothelioma cell lines, but not the NF2 wild-type HEK293 cells, is suppressed by NF2 overexpression. B) Effect of SWTX-143 on the proliferation of the Hippo-wild type mesothelioma cell line H2062.

Figure S3 shows Heatmap of differential pathway analysis comparing the tumors of vehicle and SWTX-143-treated mice.

Figure S4 shows A) Immunofluorescence stainings of lung sections from mice with mesothelioma after 2 weeks after Adeno-Cre injection, stained for Vimentin (green) and Mesothelin (red). B) Picture of an H&E staining of a lung section from a mouse with mesothelioma that was treated with SWTX-143 for one week.

Dengue is a global public health threat, with about half of the world's population at risk of contracting this mosquito-borne viral disease. Climate change, urbanization, and global travel accelerate the spread of dengue virus (DENV) to new areas, including southern parts of Europe and the US. Currently, no dengue-specific small-molecule antiviral for prophylaxis or treatment is available. Here, we report the discovery of JNJ-1802 as a potent, pan-serotype DENV inhibitor (EC50's ranging from 0.057 to 11 nM against the four DENV serotypes). The observed oral bioavailability of JNJ-1802 across preclinical species, its low clearance in human hepatocytes, the absence of major in vitro pharmacology safety alerts, and a dose-proportional increase in efficacy against DENV-2 infection in mice were all supportive of its selection as a development candidate against dengue. JNJ-1802 is being progressed in clinical studies for the prevention or treatment of dengue.

Rabies, a viral zoonosis, is responsible for almost 59,000 deaths each year, despite the existence of an effective post-exposure prophylaxis. Indeed, rabies causes acute encephalomyelitis, with a case-fatality rate of 100 % after the onset of neurological clinical signs. Therefore, the development of therapies to inhibit the rabies virus (RABV) is crucial. Here, we identified, from a 30,000 compound library screening, phthalazinone derivative compounds as potent inhibitors of RABV infection and more broadly of Lyssavirus and even Mononegavirales infections. Combining in vitro experiments, structural modelling, in silico docking and in vivo assays, we demonstrated that phthalazinone derivatives display a strong inhibition of lyssaviruses infection by acting directly on the replication complex of the virus, and with noticeable effects in delaying the onset of the clinical signs in our mouse model.

Assess in vitro activity and in vivo efficacy and safety of BHV-2100.

Figure S2 shows A) Heatmap showing YAP signature genes in vehicle and SWTX-143-treated kidney cancer cell line. B) Gene Set Enrichment Analysis of the YAP signature.

Abstract The Hippo pathway and its downstream effectors, the YAP and TAZ transcriptional coactivators, are deregulated in multiple different types of human cancer and are required for cancer cell phenotypes in vitro and in vivo, while largely dispensable for tissue homeostasis in adult mice. YAP/TAZ and their main partner transcription factors, the TEAD1–4 factors, are therefore promising anticancer targets. Because of frequent YAP/TAZ hyperactivation caused by mutations in the Hippo pathway components NF2 and LATS2, mesothelioma is one of the prime cancer types predicted to be responsive to YAP/TAZ-TEAD inhibitor treatment. Mesothelioma is a devastating disease for which currently no effective treatment options exist. Here, we describe a novel covalent YAP/TAZ-TEAD inhibitor, SWTX-143, that binds to the palmitoylation pocket of all four TEAD isoforms. SWTX-143 caused irreversible and specific inhibition of the transcriptional activity of YAP/TAZ-TEAD in Hippo-mutant tumor cell lines. More importantly, YAP/TAZ-TEAD inhibitor treatment caused strong mesothelioma regression in subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model. Finally, SWTX-143 also selectively impaired the growth of NF2-mutant kidney cancer cell lines, suggesting that the sensitivity of mesothelioma models to these YAP/TAZ-TEAD inhibitors can be extended to other tumor types with aberrations in Hippo signaling. In brief, we describe a novel and specific YAP/TAZ-TEAD inhibitor that has potential to treat multiple Hippo-mutant solid tumor types.

Nucleocytoplasmatic transport plays an essential role in eukaryotic cell homeostasis and is mediated by karyopherins. Importin β1 (KPNB1) and its adaptor protein importin α1 (KPNA2) are the best-characterized karyopherins that effect nuclear import. Here, we identify a novel small-molecule inhibitor of the importin β1-mediated nuclear import. We design a reporter cell line by stably tagging endogenous importin α1 with a fluorescent protein to screen for compounds affecting its subcellular localization. We identify a series of compounds that trigger cytoplasmatic accumulation of importin α1. The lead compound, ibetazol, is further characterized in a broad sequence of cellular nuclear transport assays. Ibetazol is shown to inhibit all importin β1-mediated nuclear import quickly and specifically, without affecting transport mediated by other karyopherins. Detailed molecular mechanism of action studies demonstrate that ibetazol inhibits importin β1 by covalently targeting Cys585. In summary, ibetazol is a novel small molecule inhibitor of importin β1 enabling pharmacological inhibition of the importin β1-mediated nuclear import process with wide applicability in different fields.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the β2-adrenergic receptor ( β 2 -AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the β 2 -AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I 125 -cyanopindolol, a β 2 -AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a β 2 -AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the β 2 -AR. Oral administration of glucose with a non-bioavailable β 2 -AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical β 2 -ARs in stimulating gut glucose uptake, and suggesting enterocyte β 2 -AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether β 2 -ARs mediate glucose sensing also in other tissues.