Structural basis of Δ9-THC analog activity at the Cannabinoid 1 receptor.

Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC's psychoactive actions are mediated primarily by the cannabinoid receptor CB1. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB1 and its primary transducer, Gi1. We leverage this structure for docking and 1,000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of Gi and ß-arrestins and reversibility of binding from an active complex. By combining detailed CB1 structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Targeting NOX2 with Bivalent Small-Molecule p47phox-p22phox Inhibitors.

Nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) is an enzymatic complex whose function is the regulated generation of reactive oxygen species (ROS). NOX2 activity is central to redox signaling events and antibacterial response, but excessive ROS production by NOX2 leads to oxidative stress and inflammation in a range of diseases. The protein-protein interaction between the NOX2 subunits p47phox and p22phox is essential for NOX2 activation, thus p47phox is a potential drug target. Previously, we identified 2-aminoquinoline as a fragment hit toward p47phoxSH3A-B and converted it to a bivalent small-molecule p47phox-p22phox inhibitor (Ki = 20 µM). Here, we systematically optimized the bivalent compounds by exploring linker types and positioning as well as substituents on the 2-aminoquinoline part and characterized the bivalent binding mode with biophysical methods. We identified several compounds with submicromolar binding affinities and cellular activity and thereby demonstrated that p47phox can be targeted by potent small molecules.

Realizing integration in structural biology: The 2022 ISBUC Annual Meeting.

This meeting report presents the 2022 Annual Meeting of the cluster for Integrative Structural Biology at the University of Copenhagen (ISBUC) and discusses the cluster approach to interdisciplinary research management. This approach successfully facilitates cross-faculty and inter-departmental collaboration. Innovative integrative research collaborations ignited by ISBUC, as well as research presented at the meeting, are showcased.

Small-molecule modulators of tumor necrosis factor signaling.

Tumor necrosis factor (TNF) is a pleiotropic cytokine with a major role in immune system homeostasis and is involved in many inflammatory and autoimmune diseases, such as rheumatoid arthritis (RA), psoriasis, Alzheimer's disease (AD), and multiple sclerosis (MS). Thus, TNF and its receptors, TNFR1 and TNFR2, are relevant pharmacological targets. Biologics have been developed to block TNF-dependent signaling cascades, but they display serious side effects, and their pharmacological effectiveness decreases over time because of their immunogenicity. In this review, we present recent discoveries in small molecules targeting TNF and its receptors and discuss alternative strategies for modulating TNF signaling.

Development of Noncovalent Small-Molecule Keap1-Nrf2 Inhibitors by Fragment-Based Drug Discovery.

Targeting the protein-protein interaction (PPI) between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its repressor, Kelch-like ECH-associated protein 1 (Keap1), constitutes a promising strategy for treating diseases involving oxidative stress and inflammation. Here, a fragment-based drug discovery (FBDD) campaign resulted in novel, high-affinity (Ki = 280 nM), and cell-active noncovalent small-molecule Keap1-Nrf2 PPI inhibitors. We screened 2500 fragments using orthogonal assays─fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)─and validated the hits by saturation transfer difference (STD) NMR, leading to 28 high-priority hits. Thirteen co-structures showed fragments binding mainly in the P4 and P5 subpockets of Keap1's Kelch domain, and three fluorenone-based fragments featuring a novel binding mode were optimized by structure-based drug discovery. We thereby disclose several fragment hits, including their binding modes, and show how FBDD can be performed to find new small-molecule Keap1-Nrf2 PPI inhibitors.

Structure of the proteolytic enzyme PAPP-A with the endogenous inhibitor stanniocalcin-2 reveals its inhibitory mechanism.

The metzincin metalloproteinase PAPP-A plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). Using single-particle cryo-electron microscopy (cryo-EM), we here report the structure of PAPP-A in complex with its endogenous inhibitor, stanniocalcin-2 (STC2), neither of which have been reported before. The highest resolution (3.1 Å) was obtained for the STC2 subunit and the N-terminal approximately 1000 residues of the PAPP-A subunit. The 500 kDa 2:2 PAPP-A·STC2 complex is a flexible multidomain ensemble with numerous interdomain contacts. In particular, a specific disulfide bond between the subunits of STC2 and PAPP-A prevents dissociation, and interactions between STC2 and a module located in the very C-terminal end of the PAPP-A subunit prevent binding of its main substrate, IGFBP-4. While devoid of activity towards IGFBP-4, the active site cleft of the catalytic domain is accessible in the inhibited PAPP-A·STC2 complex, as shown by its ability to hydrolyze a synthetic peptide derived from IGFBP-4. Relevant to multiple human pathologies, this unusual mechanism of proteolytic inhibition may support the development of specific pharmaceutical agents, by which IGF signaling can be indirectly modulated.

Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Reversal of ABCG2/BCRP-Mediated Multidrug Resistance by 5,3',5'-Trihydroxy-3,6,7,4'-Tetramethoxyflavone Isolated from the Australian Desert Plant Eremophila galeata Chinnock.

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3',5'-trihydroxy-3,6,7,4'-tetramethoxyflavone (2), which at a concentration of 25 µg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 µM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

Deconstructing Noncovalent Kelch-like ECH-Associated Protein 1 (Keap1) Inhibitors into Fragments to Reconstruct New Potent Compounds.

Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220-380-fold stronger affinity (Ki = 16 µM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04-0.5 µM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.

Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.

Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65 Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).

Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles.

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27 Specificity.

KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyze demethylation of di- and trimethylated lysine on histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS), the enzymes do not catalyze demethylation of this general marker of repression. To address this question for KDM6B, we used computational methods to identify H3(17-33)-derived peptides with improved binding affinity that would allow co-crystallization with the catalytic core of human KDM6B (ccKDM6B). A total of five peptides were identified, and their IC50 values were determined in a matrix-assisted laser desorption ionization time-of-flight-based assay. Despite none of the peptides showing affinity significantly higher than that of the H3(17-33) peptide, it was possible to co-crystallize ccKDM6B with a H3(17-33)A21M peptide. This structure reveals the interactions between the KDM6B zinc binding domain and the H3(17-23) region. Although KDM6A and KDM6B differ in primary sequence, particularly in the H3L20 binding pocket of the zinc binding domains, where two histidines in KDM6A have been replaced by a glutamate and a tyrosine, they bind H3(17-23) in a very similar fashion. This structure shows that KDM6B, in analogy with KDM6A, also uses the zinc binding domain to achieve H3K27me3/me2 specificity. The histidine to glutamine substitution at amino acid position 1564 in the KDM6B zinc binding domain can further explain why KDM6B binds substrates with an affinity higher than that of KDM6A.

Lysine demethylase inhibition protects pancreatic ß cells from apoptosis and improves ß-cell function.

Transcriptional changes control ß-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs) protects ß cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate ß-cell fate in response to inflammatory stress. Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting a possible role in inflammation-induced ß-cell destruction. Inhibition of KDM6 demethylases using the selective inhibitor GSK-J4 protected insulin-producing cells and human and mouse islets from cytokine-induced apoptosis by blunting nuclear factor (NF)-κB signaling and endoplasmic reticulum (ER) stress response gene expression. GSK-J4 furthermore increased expression of insulin gene and glucose-stimulated insulin secretion. Expression of genes regulating purinergic and cytokine ligand-receptor interactions was downregulated following GSK-J4 exposure, while expression of genes involved in cell maintenance and survival was upregulated. These data suggest that KDMs are important regulators of inflammation-induced ß-cell dysfunction and death.

The prototypical proton-coupled oligopeptide transporter YdgR from Escherichia coli facilitates chloramphenicol uptake into bacterial cells.

Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS-based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Structure-Based Design of a New Scaffold for Cell-Penetrating Peptidic Inhibitors of the Histone Demethylase PHF8.

The histone demethylase PHF8 catalyzes demethylation of mono- and di-methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri-methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri-methylated Lys4 was used as a template for the structure-based design of a cyclic, cell-penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free N terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptide's ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.