Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

Predicting the target landscape of kinase inhibitors using 3D convolutional neural networks.

Many therapies in clinical trials are based on single drug-single target relationships. To further extend this concept to multi-target approaches using multi-targeted drugs, we developed a machine learning pipeline to unravel the target landscape of kinase inhibitors. This pipeline, which we call 3D-KINEssence, uses a new type of protein fingerprints (3D FP) based on the structure of kinases generated through a 3D convolutional neural network (3D-CNN). These 3D-CNN kinase fingerprints were matched to molecular Morgan fingerprints to predict the targets of each respective kinase inhibitor based on available bioactivity data. The performance of the pipeline was evaluated on two test sets: a sparse drug-target set where each drug is matched in most cases to a single target and also on a densely-covered drug-target set where each drug is matched to most if not all targets. This latter set is more challenging to train, given its non-exclusive character. Our model's root-mean-square error (RMSE) based on the two datasets was 0.68 and 0.8, respectively. These results indicate that 3D FP can predict the target landscape of kinase inhibitors at around 0.8 log units of bioactivity. Our strategy can be utilized in proteochemometric or chemogenomic workflows by consolidating the target landscape of kinase inhibitors.

PSW-Designer: An Open-Source Computational Platform for the Design and Virtual Screening of Photopharmacological Ligands.

Photoswitchable (PSW) molecules offer an attractive opportunity for the optical control of biological processes. However, the successful design of such compounds remains a challenging multioptimization endeavor, resulting in several biological target classes still relatively poorly explored by photoswitchable ligands, as is the case for G protein-coupled receptors (GPCRs). Here, we present the PSW-Designer, a fully open-source computational platform, implemented in the KNIME Analytics Platform, to design and virtually screen novel photoswitchable ligands for photopharmacological applications based on privileged scaffolds. We demonstrate the applicability of the PSW-Designer to GPCRs and assess its predictive capabilities via two retrospective case studies. Furthermore, by leveraging bioactivity information on known ligands, typical and atypical strategies for photoswitchable group incorporation, and the increasingly structural information available for biological targets, the PSW-Design will facilitate the design of novel photoswitchable molecules with improved photopharmacological properties and increased binding affinity shifts upon illumination for GPCRs and many other protein targets.

KLIFS: an overhaul after the first 5 years of supporting kinase research.

Kinases are a prime target of drug development efforts with >60 drug approvals in the past two decades. Due to the research into this protein family, a wealth of data has been accumulated that keeps on growing. KLIFS-Kinase-Ligand Interaction Fingerprints and Structures-is a structural database focusing on how kinase inhibitors interact with their targets. The aim of KLIFS is to support (structure-based) kinase research through the systematic collection, annotation, and processing of kinase structures. Now, 5 years after releasing the initial KLIFS website, the database has undergone a complete overhaul with a new website, new logo, and new functionalities. In this article, we start by looking back at how KLIFS has been used by the research community, followed by a description of the renewed KLIFS, and conclude with showcasing the functionalities of KLIFS. Major changes include the integration of approved drugs and inhibitors in clinical trials, extension of the coverage to atypical kinases, and a RESTful API for programmatic access. KLIFS is available at the new domain https://klifs.net.

SAR exploration of the non-imidazole histamine H3 receptor ligand ZEL-H16 reveals potent inverse agonism.

Histamine H3 receptor (H3 R) agonists without an imidazole moiety remain very scarce. Of these, ZEL-H16 (1) has been reported previously as a high-affinity non-imidazole H3 R (partial) agonist. Our structure-activity relationship analysis using derivatives of 1 identified both basic moieties as key interaction motifs and the distance of these from the central core as a determinant for H3 R affinity. However, in spite of the reported H3 R (partial) agonism, in our hands, 1 acts as an inverse agonist for Gαi signaling in a CRE-luciferase reporter gene assay and using an H3 R conformational sensor. Inverse agonism was also observed for all of the synthesized derivatives of 1. Docking studies and molecular dynamics simulations suggest ionic interactions/hydrogen bonds to H3 R residues D1143.32 and E2065.46 as essential interaction points.

To Target or Not to Target Schistosoma mansoni Cyclic Nucleotide Phosphodiesterase 4A?

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

Green Drug Discovery: Novel Fragment Space from the Biomass-Derived Molecule Dihydrolevoglucosenone (CyreneTM).

Biomass-derived molecules can provide a basis for sustainable drug discovery. However, their full exploration is hampered by the dominance of millions of old-fashioned screening compounds in classical high-throughput screening (HTS) libraries frequently utilized. We propose a fragment-based drug discovery (FBDD) approach as an efficient method to navigate biomass-derived drug space. Here, we perform a proof-of-concept study with dihydrolevoglucosenone (CyreneTM), a pyrolysis product of cellulose. Diverse synthetic routes afforded a 100-membered fragment library with a diversity in functional groups appended. The library overall performs well in terms of novelty, physicochemical properties, aqueous solubility, stability, and three-dimensionality. Our study suggests that Cyrene-based fragments are a valuable green addition to the drug discovery toolbox. Our findings can help in paving the way for new hit drug candidates that are based on renewable resources.

Progress in Free Energy Perturbation: Options for Evolving Fragments.

One of the remaining bottlenecks in fragment-based drug design (FBDD) is the initial exploration and optimization of the identified hit fragments. There is a growing interest in computational approaches that can guide these efforts by predicting the binding affinity of newly designed analogues. Among others, alchemical free energy (AFE) calculations promise high accuracy at a computational cost that allows their application during lead optimization campaigns. In this review, we discuss how AFE could have a strong impact in fragment evolution, and we raise awareness on the challenges that could be encountered applying this methodology in FBDD studies.

Escape from planarity in fragment-based drug discovery: A physicochemical and 3D property analysis of synthetic 3D fragment libraries.

Fragment-based drug discovery (FBDD) has grown into a well-established approach in the pursuit of new therapeutics. Key to the success of FBDD is the low molecular complexity of the initial hits and this has resulted in fragment libraries that mainly contain compounds with a two-dimensional (2D) shape. In an effort to increase the chemical diversity and explore the impact of increased molecular complexity on the hit rate of fragment library screening, several academic and industrial groups have designed and synthesised novel fragments with a three-dimensional (3D) shape. This review provides an overview of 25 synthetic 3D fragment libraries from the recent literature. We calculate and compare physicochemical properties and descriptors that are typically used to measure molecular three-dimensionality such as fraction sp3 (Fsp3), plane of best fit (PBF) scores and principal moment of inertia (PMI) plots. Although the libraries vary widely in structure and properties, some key common features can be identified which may have utility in designing the next generation of 3D fragment libraries.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2).

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors.

Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.

Covalent Inhibition of the Histamine H3 Receptor.

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.

A Photoswitchable Agonist for the Histamine H3 Receptor, a Prototypic Family A G-Protein-Coupled Receptor.

Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H3 receptor (hH3 R). Upon illumination, key compound 65 decreases its affinity for the hH3 R by 8.5-fold and its potency in hH3 R-mediated Gi protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH3 R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.

A toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with light.

We report a detailed structure-activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle efficacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling.

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology.

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/ cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling.

KLIFS: a structural kinase-ligand interaction database.

Protein kinases play a crucial role in cell signaling and are important drug targets in several therapeutic areas. The KLIFS database contains detailed structural kinase-ligand interaction information derived from all (>2900) structures of catalytic domains of human and mouse protein kinases deposited in the Protein Data Bank in order to provide insights into the structural determinants of kinase-ligand binding and selectivity. The kinase structures have been processed in a consistent manner by systematically analyzing the structural features and molecular interaction fingerprints (IFPs) of a predefined set of 85 binding site residues with bound ligands. KLIFS has been completely rebuilt and extended (>65% more structures) since its first release as a data set, including: novel automated annotation methods for (i) the assessment of ligand-targeted subpockets and the analysis of (ii) DFG and (iii) αC-helix conformations; improved and automated protocols for (iv) the generation of sequence/structure alignments, (v) the curation of ligand atom and bond typing for accurate IFP analysis and (vi) weekly database updates. KLIFS is now accessible via a website (http://klifs.vu-compmedchem.nl) that provides a comprehensive visual presentation of different types of chemical, biological and structural chemogenomics data, and allows the user to easily access, compare, search and download the data.

Photoswitching the Efficacy of a Small-Molecule Ligand for a Peptidergic GPCR: from Antagonism to Agonism.

For optical control of GPCR function, we set out to develop small-molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene-containing CXCR3 ligands. G protein activation assays and real-time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron-donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling.

3D-e-Chem-VM: Structural Cheminformatics Research Infrastructure in a Freely Available Virtual Machine.

3D-e-Chem-VM is an open source, freely available Virtual Machine ( http://3d-e-chem.github.io/3D-e-Chem-VM/ ) that integrates cheminformatics and bioinformatics tools for the analysis of protein-ligand interaction data. 3D-e-Chem-VM consists of software libraries, and database and workflow tools that can analyze and combine small molecule and protein structural information in a graphical programming environment. New chemical and biological data analytics tools and workflows have been developed for the efficient exploitation of structural and pharmacological protein-ligand interaction data from proteomewide databases (e.g., ChEMBLdb and PDB), as well as customized information systems focused on, e.g., G protein-coupled receptors (GPCRdb) and protein kinases (KLIFS). The integrated structural cheminformatics research infrastructure compiled in the 3D-e-Chem-VM enables the design of new approaches in virtual ligand screening (Chemdb4VS), ligand-based metabolism prediction (SyGMa), and structure-based protein binding site comparison and bioisosteric replacement for ligand design (KRIPOdb).

The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity.

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQp, FtsBp, and FtsLp) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQp, FtsBp, and FtsLp individually and in combination. Upon co-expression, FtsQp was co-purified with FtsBp and FtsLp from E. coli extracts as a stable trimeric complex. FtsBp was also shown to interact with FtsQp in the absence of FtsLp albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQpBpLp complex and the FtsQpBp subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases.

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.