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A conference paper is presented which offers information on SFCscore, a collection of empirical scoring functions derived from a set of over 60 descriptors for protein-ligand complexes of known structure and also discusses an affinity prediction model for the complexes. It presents a technique which uses the software Random Forests to derive a new function (SFCscoreRF) based on the SFCscore descriptors as input data.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi , Burkholderia pseudomallei , and Legionella pneumophila , express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta -halogenated phenyl sulfonamide to be responsible for the high affinity.

Covalent inhibition with electrophiles of suitable reactivity allows for prolonged inactivation of a targeted enzyme, in comparison to noncovalent inhibitors. To fine-tune covalently reacting groups, various computational workflows have been developed for reactivity prediction. Their results are typically evaluated based on in vitro assays that use cysteine, glutathione, or other model nucleophiles, but the relevance of the predictions for the situation in a particular enzyme environment remains under-investigated. Herein, we utilized a DFT-based computational method to assess the transferability of predicted reactivities to enzyme inhibition. For this, we designed and synthesized a set of covalent inhibitors of ChlaDUB1, a potential target to fight the pathogen Chlamydia trachomatis . We show that in the context of enzyme inhibition, geometrical congruence between ligands has to be taken into account when evaluating the reactivity of covalently reacting groups in respective drug discovery projects., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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 IC 50 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 development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets., (© 2024. The Author(s).)

Fragment-based screening has become indispensable in drug discovery. Yet, the weak binding affinities of these small molecules still represent a challenge for the reliable detection of fragment hits. The extent of this issue was illustrated in the literature for the aspartic protease endothiapepsin: When seven biochemical and biophysical in vitro screening methods were applied to screen a library of 361 fragments, very poor overlap was observed between the hit fragments identified by the individual approaches, resulting in high levels of false positive and/or false negative results depending on the mutually compared methods. Here, the reported in vitro findings are juxtaposed with the results from in silico docking and scoring approaches. The docking programs GOLD and Glide were considered with the scoring functions ASP, ChemScore, ChemPLP, GoldScore, DSX CSD , and GlideScore. First, the ranking power and scoring power were assessed for the named scoring functions. Second, the capability of reproducing the crystallized fragment binding modes was tested in a structure-based redocking approach. The redocking success notably depended on the ligand efficiency of the considered fragments. Third, a blinded virtual screening approach was employed to evaluate whether in silico screening can compete with in vitro methods in the enrichment of fragment databases., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

The covalent modification of proteins with polymers is a well-established method for improving the pharmacokinetic properties of therapeutically valuable biologics. The conjugated polymer chains of the resulting hybrid represent highly flexible macromolecular structures. As the dynamics of such systems remain rather elusive for established experimental techniques from the field of protein structure elucidation, molecular dynamics simulations have proven as a valuable tool for studying such conjugates at an atomistic level, thereby complementing experimental studies. With a focus on new developments, this review aims to provide researchers from the polymer bioconjugation field with a concise and up to date overview of such approaches. After introducing basic principles of molecular dynamics simulations, as well as methods for and potential pitfalls in modeling bioconjugates, the review illustrates how these computational techniques have contributed to the understanding of bioconjugates and bioconjugation strategies in the recent past and how they may lead to a more rational design of novel bioconjugates in the future.

A sufficiently stable noncovalent association complex between a covalent inhibitor and its protein target is regarded as a prerequisite for the formation of a covalent complex. As this transient form can hardly be assessed experimentally, computational modeling is required to probe the suitability of a given ligand at this particular stage. To investigate which criteria should be fulfilled by suitable candidates in a molecular dynamics (MD) assessment, a systematic study was conducted with 20 complexes of cathepsin K, a papain-like cysteine protease of pharmaceutical relevance. The covalent inhibitors in these complexes were converted to their pre-reaction states, and the resulting noncovalent complexes were subjected to MD simulations. The critical distance between the electrophilic and nucleophilic reaction partners was monitored as a potential parameter to assess the suitability for covalent bond formation. Across various warhead types, a distance between 3.6 and 4.0 Å, comparable to the sum of the generalized Born radii of carbon and sulfur, was found to be stably maintained under appropriate conditions. The protonation state of the catalytic dyad and the resulting solvation pattern dramatically affected the noncovalent binding mode and the distance of the warhead to the active site. For several complexes, fluctuations in the orientation of the warhead were observed due to torsional rotations in adjacent bonds. This observation helped to explain the gradual transitions from noncovalent to covalent complexes observed in the crystal structures of three closely related nitrile-based inhibitors. According to comparative simulations conducted for a set of 13 cathepsin S complexes, the overall findings of the study appear to be transferable to related cysteine proteases as targets of covalent inhibitors.

Polymer self-assembly leading to cooling-induced hydrogel formation is relatively rare for synthetic polymers and typically relies on H-bonding between repeat units. Here, we describe a non-H-bonding mechanism for a cooling-induced reversible order-order (sphere-to-worm) transition and related thermogelation of solutions of polymer self-assemblies. A multitude of complementary analytical tools allowed us to reveal that a significant fraction of the hydrophobic and hydrophilic repeat units of the underlying block copolymer is in close proximity in the gel state. This unusual interaction between hydrophilic and hydrophobic blocks reduces the mobility of the hydrophilic block significantly by condensing the hydrophilic block onto the hydrophobic micelle core, thereby affecting the micelle packing parameter. This triggers the order-order transition from well-defined spherical micelles to long worm-like micelles, which ultimately results in the inverse thermogelation. Molecular dynamics modeling indicates that this unexpected condensation of the hydrophilic corona onto the hydrophobic core is due to particular interactions between amide groups in the hydrophilic repeat units and phenyl rings in the hydrophobic ones. Consequently, changes in the structure of the hydrophilic blocks affecting the strength of the interaction could be used to control macromolecular self-assembly, thus allowing for the tuning of gel characteristics such as strength, persistence, and gelation kinetics. We believe that this mechanism might be a relevant interaction pattern for other polymeric materials as well as their interaction in and with biological environments. For example, controlling the gel characteristics could be considered important for applications in drug delivery or biofabrication.

Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A-MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A-MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders., (open access.)

Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97., (© 2022. The Author(s).)

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates., (© 2022. The Author(s).)

Activation of the human cannabinoid receptor type 1 ( h CB 1 R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for h CB 1 R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB 1 R ( K i ( cis- form) = 0.18 μM), with a marked difference in affinity with respect to its inactive " trans -off" form (CB 1 R K i trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT βarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of h CB 1 R in disorders associated with the endocannabinoid system.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k 2nd  = 883 • 10 3  M -1  s -1 ) with single-digit nanomolar binding affinity (K i  = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Predicting biopharmaceutical characteristics and food effects for drug substances may substantially leverage rational formulation outcomes. We established a bile and lipid interaction prediction model for new drug substances and further explored the model for the prediction of bile-related food effects. One hundred and forty-one drugs were categorized as bile and/or lipid interacting and noninteracting drugs using 1 H nuclear magnetic resonance (NMR) spectroscopy. Quantitative structure-property relationship modeling with molecular descriptors was applied to predict a drug's interaction with bile and/or lipids. Bile interaction, for example, was indicated by two descriptors characterizing polarity and lipophilicity with a high balanced accuracy of 0.8. Furthermore, the predicted bile interaction correlated with a positive food effect. Reliable prediction of drug substance interaction with lipids required four molecular descriptors with a balanced accuracy of 0.7. These described a drug's shape, lipophilicity, aromaticity, and hydrogen bond acceptor capability. In conclusion, reliable models might be found through drug libraries characterized for bile interaction by NMR. Furthermore, there is potential for predicting bile-related positive food effects.

To develop tools to investigate the biological functions of butyrylcholinesterase (BChE) and the mechanisms by which BChE affects Alzheimer's disease (AD), we synthesized several selective, nanomolar active, pseudoirreversible photoswitchable BChE inhibitors. The compounds were able to specifically influence different kinetic parameters of the inhibition process by light. For one compound, a 10-fold difference in the IC 50 -values (44.6 nM cis , 424 nM trans ) in vitro was translated to an "all or nothing" response with complete recovery in a murine cognition-deficit AD model at dosages as low as 0.3 mg/kg.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABA A receptors (GABA A Rs). In this study, the modulatory potential of 11 SQTs at GABA A Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of β-caryophyllene and α-humulene, as well as their respective derivatives β-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABA A R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ 2 and δ subunits is important for SQT modulation. While phasic GABA A receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α 1 β 2 γ 2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABA A R localized between transmembrane segments 1 and 3 at the ( + α)-( - α) interface. In sum, differences in the modulation of GABA A R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

Human cannabinoid receptor type 1 ( h CB 1 R) plays important roles in the regulation of appetite and development of addictive behaviors. Herein, we describe the design, synthesis, photocharacterization, molecular docking, and in vitro characterization of "photo-rimonabant", i.e., azo-derivatives of the selective h CB 1 R antagonist SR1411716A (rimonabant). By applying azo-extension strategies, we yielded compound 16a , which shows marked affinity for CB 1 R ( K i ( cis  form) = 29 nM), whose potency increases by illumination with ultraviolet light (CB 1 R K i trans / cis ratio = 15.3). Through radioligand binding, calcium mobilization, and cell luminescence assays, we established that 16a is highly selective for h CB 1 R over h CB 2 R. These selective antagonists can be valuable molecular tools for optical modulation of CBRs and better understanding of disorders associated with the endocannabinoid system.

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis , InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu 2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aβ25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a , with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL pro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL pro . Moreover, we report the discovery of isoindolines as a new class of potent PL pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL pro are valuable starting points for the development of new pan-coronaviral inhibitors., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Melatonin MT 1 and MT 2 receptor ligands have been vigorously explored for the last 4 decades. Inspection of approximately 80 publications in the field revealed that most melatonergic ligands were structural analogues of melatonin combining three essential features of the parent compound: an aromatic ring bearing a methoxy group and an amide side chain in a relative arrangement similar to that present in melatonin. While several series of MT 2 -selective agents-agonists, antagonists, or partial agonists-were reported, the field was lacking MT 1 -selective agents. Herein, we describe various approaches toward the development of melatonergic ligands, keeping in mind that most of the molecules/pharmacophores obtained were essentially melatonin copies, even though diverse tri- or tetra-cyclic compounds were explored. In addition to lack of structural diversity, only few studies examined the activity of the reported melatonergic ligands in vivo. Moreover, an extensive pharmacological characterization including biopharmaceutical stability, pharmacokinetic properties, specificity toward other major receptors to name a few remained scarce. For example, many of the antagonists described were not stable in vivo, were not selective for the melatonin receptor subtype of interest, and were not fully characterized from a pharmacological standpoint. Indeed, virtual screening of large compound libraries has led to the recent discovery of potent and selective melatonin receptor agonists and partial agonists of new chemotypes. Having said this, the melatonergic field is still lacking subtype-selective melatonin receptor antagonists "active" in vivo, which are critical to our understanding of melatonin and melatonin receptors' role in basic physiology and disease., (© 2020 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

A model system of two related enzymes with conserved binding sites, namely N -myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M 1 /M 4 -preferring orthosteric muscarinic agonist, with that of tacrine, a well-known acetylcholinesterase (AChE) inhibitor able to allosterically modulate muscarinic acetylcholine receptors (mAChRs). When tested in vitro in a colorimetric assay for their ability to inhibit AChE, the new compounds showed higher or similar potency compared to that of tacrine. Docking analyses were performed on the most potent inhibitors in the series (8-C8, 8-C10, 8-C12) to rationalize their experimental inhibitory power against AChE. Next, we evaluated the signaling cascade at M 1 mAChRs by exploring the interaction of Gα q -PLC-β3 proteins through split luciferase assays and the myo-Inositol 1 phosphate (IP1) accumulation in cells. The results were compared with those obtained on the known derivatives 6-C7 and 6-C10, two quite potent AChE inhibitors in which tacrine is linked to iperoxo, an exceptionally potent muscarinic orthosteric activator. Interestingly, we found that 6-C7 and 6-C10 behaved as partial agonists of the M 1 mAChR, at variance with hybrids 7-Cn and 8-Cn containing xanomeline as the orthosteric molecular fragment, which were all unable to activate the receptor subtype response., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for h BChE over human acetycholinesterase ( h AChE), yielding short-, medium-, and long-acting nanomolar h BChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer's disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD.

(11 S )-11-Aminostrychnine ( 1 ) and N -[(11 S )-strychnine-11-yl]propionamide ( 2 ) were synthesized and characterized as antagonists of homomeric α1 and heteromeric α1β glycine receptors in a functional fluorescence-based assay and a patch-clamp assay and in radioligand binding studies. The absolute configuration at C-11 of 1 was determined based on vicinal coupling constants and NOESY data. Docking experiments to the orthosteric binding site of the α3 glycine receptor showed a binding mode of compound 2 analogous to that of strychnine, explaining its high antagonistic potency. The findings identify the C-11 amide function of strychnine as a suitable linker group for the future development of dimeric strychnine analogues targeting glycine receptors. The findings extend the SAR of strychnine at glycine receptors.

Ubiquitin-conjugating enzymes (E2s) govern key aspects of ubiquitin signaling. Emerging evidence suggests that the activities of E2s are modulated by posttranslational modifications; the structural underpinnings, however, are largely unclear. Here, we unravel the structural basis and mechanistic consequences of a conserved autoubiquitination event near the catalytic center of E2s, using the human anaphase-promoting complex/cyclosome-associated UBE2S as a model system. Crystal structures we determined of the catalytic ubiquitin carrier protein domain combined with MD simulations reveal that the active-site region is malleable, which permits an adjacent ubiquitin acceptor site, Lys +5 , to be ubiquitinated intramolecularly. We demonstrate by NMR that the Lys +5 -linked ubiquitin inhibits UBE2S by obstructing its reloading with ubiquitin. By immunoprecipitation, quantitative mass spectrometry, and siRNA-and-rescue experiments we show that Lys +5 ubiquitination of UBE2S decreases during mitotic exit but does not influence proteasomal turnover of this E2. These findings suggest that UBE2S activity underlies inherent regulation during the cell cycle., (Copyright © 2019 Elsevier Ltd. All rights reserved.)