Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.

The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that the human endolysosomal cation channel TRPML2 agonist ML2-SA1 impairs the late life cycle stages of ZIKV, thus underscoring TRPML2 as a promising antiviral target. Upon treatment with ML2-SA1, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced. ML2-SA1-treated cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound’s negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV as well as many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed ML2-SA1 to also negatively impact HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides underscoring the potential of TRPML2 as a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.### Competing Interest StatementThe authors have declared no competing interest.* aa : amino acids cLSM : confocal laser scanning microscopy CTCF : corrected total cell fluorescence CV : coefficient of variation DENV : Dengue virus DMSO : dimethyl sulfoxide E : protein envelope protein FBS : fetal bovine serum GEs : genome equivalents HCV : Hepatitis C virus HEV : Hepatitis E virus hpi : hours post infection IAV : Influenza A virus ML2-SA1 : mucolipin 2 synthetic agonist 1 ML-SA1 : mucolipin synthetic agonist 1 MOI : multiplicity of infection MVB : multivesicular body PBS : phosphate buffered saline PFU : plaque forming units PIFA : (bis(trifluoroacetoxy)iodo)benzene PMSF : phenylmethylsulfonyl fluoride Renilla : Luciferase R Luc RIPA : radio-immunoprecipitation assay rt : room temperature RT-qPCR : real-time quantitative polymerase chain reaction SD : standard deviation STED : stimulated emission depletion TBS : tris buffered saline TCID50 : half maximal tissue culture infective dose tMOC : thresholded Mender’s overlap coefficient TPC : two-pore channels TRPML : transient receptor potential mucolipin WNV : West Nile virus YFV : yellow fever virus ZIKV : FP ZIKV French Polynesia isolate ZIKV : U ZIKV Uganda isolate ZIKV : Zika virus

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 μM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 μM, 68% at 1 μM, 35% at 0.1 μM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Host ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that treatment with ML2-SA1, an agonist for the human endolysosomal cation channel TRPML2, impairs ZIKV replication. Upon ML2-SA1 treatment, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced and cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound's negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV and many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed that ML2-SA1 also negatively impacted HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides indicating that TRPML2 may be a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.

Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Potential (trifluoromethyl)diazirine-based TRPML1 ion channel ligands were designed and synthesized, and their structures were determined by single-crystal X-ray diffraction analysis. Photoactivation studies via 19F NMR spectroscopy and HPLC-MS analysis revealed distinct kinetical characteristics in selected solvents and favorable photochemical properties in an aqueous buffer. These photoactivatable TRPML activators represent useful and valuable tools for TRPML photoaffinity labeling combined with mass spectrometry.

TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0 angstrom) and 4.5 (2.95 angstrom), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca2+ binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca2+ concentration do not affect the protein's secondary structure, but can influence ELD oligomer integrity according to native mass spectrometry. Our data thus complete the set of high-resolution views of human TRPML channel ELDs and reveal some structural responses to the conditions the TRPML2 ELD encounters as the channel traffics through the endolysosomal system.

Four Ru-Pd heterobimetallic complexes, each one in two different coordination modes (NNSS and NS) having metals connected by a binucleating dialkyldithiooxamidate [N(R)SC-CS(R)N] [R = methyl, ethyl, n-butyl and isopropyl], were prepared by reacting the monochelate [(trinpropyl-phosphine)ClPd(HR2C2N2S2 κ-S,S-Pd)] with [(η6-p-cymene)RuCl2]2. Furthermore, two palladium homobimetallic complexes having two (trinpropyl-phosphine)ClPd moieties joined by a diethyldithiooxamidate in both κ-N,S Pd, κ-N',S' Pd' and κ-N,N' Pd, κ-S,S' Pd' coordination modes were synthesized. For both kinds of complexes, homo- and heterobimetallic, at room temperature and in chloroform solution, the NNSS coordination mode (kinetic compounds) turns out to be unstable and therefore the resulting complexes rearrange into a thermodynamically more stable form (NS coordination mode). The crystal structures of [(trinpropyl-phosphine)ClPd]2[μ-(ethyl)2-DTO κ-N,S Pd, κ-N',S' Pd'] (2) and [(η6-p-cymene)ClRu][μ-(methyl)2-DTO κ-N,S Ru, κ-N,S Pd] [(trinpropyl-phosphine)ClPd] (1c) were determined by solid state X-ray crystallography. Moreover, the higher stability of the thermodynamic species in the heterobimetallic complexes (Ru-Pd) was evaluated by means of computational studies in accordance with the maximum hardness principle. All stable NS complexes (i.e. 1c-4c, 2 and the previously reported homobimetallic Ru complex 3) were tested against two leukemia cell lines, namely the drug-sensitive CCRF-CEM cell line and its multidrug-resistant sub-cell line CEM/ADR5000 showing anti-proliferative activity in the low micromolar range (∼1-5 μM) and micromolar range (∼10-25 μM), respectively. In addition, these complexes efficaciously block at least two out of the three proteolytic activities of the tumor target 20S proteasome, with heterobimetallic complex 3c and homobimetallic complex 3 possessing the best inhibitory profile.