Comparative analysis of formyl peptide receptor 1 and formyl peptide receptor 2 reveals shared and preserved signalling profiles.

BACKGROUND AND PURPOSE: The pattern recognition receptors, formyl peptide receptors, FPR1 and FPR2, are G protein-coupled receptors that recognize many different pathogen- and host-derived ligands. While FPR1 conveys pro-inflammatory signals, FPR2 is linked with pro-resolving outcomes. To analyse how the two very similar FPRs exert opposite effects in modulating inflammatory responses despite their high homology, a shared expression profile on immune cells and an overlapping ligand repertoire, we questioned whether the signalling profile differs between these two receptors. EXPERIMENTAL APPROACH: We deduced EC50 and Emax values for synthetic, pathogen-derived and host-derived peptide agonists for both FPR1 and FPR2 and analysed them within the framework of biased signalling. We furthermore investigated whether FPR isoform-specific agonists affect the ex vivo lifespan of human neutrophils. KEY RESULTS: The FPRs share a core signature across signalling pathways. Whereas the synthetic WKYMVm and formylated peptides acted as potent agonists at FPR1, and at FPR2, only WKYMVm was a full agonist. Natural FPR2 agonists, irrespective of N-terminal formylation, displayed lower activity ratios, suggesting an underutilized signalling potential of this receptor. FPR2 agonism did not counteract LPS-induced neutrophil survival, indicating that FPR2 activation per se is not linked with a pro-resolving function. CONCLUSION AND IMPLICATIONS: Activation of FPR1 and FPR2 by a representative agonist panel revealed a lack of a receptor-specific signalling texture, challenging assumptions about distinct inflammatory profiles linked to specific receptor isoforms, signalling patterns or agonist classes. These conclusions are restricted to the specific agonists and signalling pathways examined.

Progress on the development of Class A GPCR-biased ligands.

Class A G protein-coupled receptors (GPCRs) continue to garner interest for their essential roles in cell signalling and their importance as drug targets. Although numerous drugs in the clinic target these receptors, over 60% GPCRs remain unexploited. Moreover, the adverse effects triggered by the available unbiased GPCR modulators, limit their use and therapeutic value. In this context, the elucidation of biased signalling has opened up new pharmacological avenues holding promise for safer therapeutics. Functionally selective ligands favour receptor conformations facilitating the recruitment of specific effectors and the modulation of the associated pathways. This review surveys the current drug discovery landscape of GPCR-biased modulators with a focus on recent advances. Understanding the biological effects of this preferential coupling is at different stages depending on the Class A GPCR family. Therefore, with a focus on individual GPCR families, we present a compilation of the functionally selective modulators reported over the past few years. In doing so, we dissect their therapeutic relevance, molecular determinants and potential clinical applications.

Photo-BQCA: Positive Allosteric Modulators Enabling Optical Control of the M1 Receptor.

The field of G protein-coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, i.e., photoswitchable ligands. Most of the developed tools have targeted the Rhodopsin-like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, Photo-BQCisA and Photo-BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control of the receptor using light as an external stimulus. This makes them valuable tools to further investigate M1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors.

Disrupting TSLP-TSLP receptor interactions via putative small molecule inhibitors yields a novel and efficient treatment option for atopic diseases.

Thymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein-protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.

Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Structural determinants of sphingosine-1-phosphate receptor selectivity.

Fingolimod, the prodrug of fingolimod-1-phosphate (F1P), was the first sphingosine-1-phosphate receptor (S1PR) modulator approved for multiple sclerosis. F1P unselectively targets all five S1PR subtypes. While agonism (functional antagonism via receptor internalization) at S1PR1 leads to the desired immune modulatory effects, agonism at S1PR3 is associated with cardiac adverse effects. This motivated the development of S1PR3 -sparing compounds and led to a second generation of S1PR1,5 -selective ligands like siponimod and ozanimod. Our method combines molecular dynamics simulations and three-dimensional pharmacophores (dynophores) and enables the elucidation of S1PR subtype-specific binding site characteristics, visualizing also subtle differences in receptor-ligand interactions. F1P and the endogenous ligand sphingosine-1-phosphate bind to the orthosteric pocket of all S1PRs, but show different binding mode dynamics, uncovering potential starting points for the development of subtype-specific ligands. Our study contributes to the mechanistic understanding of the selectivity profile of approved drugs like ozanimod and siponimod and pharmaceutical tool compounds like CYM5541.

Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives.

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1-4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.

Community guidelines for GPCR ligand bias: IUPHAR review 32.

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models.

Structural insights into understudied human cytochrome P450 enzymes.

Human cytochrome P450 (CYP) enzymes are widely known for their pivotal role in the metabolism of drugs and other xenobiotics as well as of endogenous chemicals. In addition, CYPs are involved in numerous pathophysiological pathways and, hence, are therapeutically relevant. Remarkably, a portion of promising CYP targets is still understudied and, as a consequence, untargeted, despite their huge therapeutic potential. An increasing number of X-ray and cryo-electron microscopy (EM) structures for CYPs have recently provided new insights into the structural basis of CYP function and potential ligand binding. This structural knowledge of CYP functionality is essential for both understanding metabolism and exploiting understudied CYPs as drug targets. In this review, we summarize and highlight structural knowledge about this enzyme class, with a focus on understudied CYPs and resulting opportunities for structure-based drug design. Teaser: This review summarizes recent structural insights into understudied cytochrome P450 enzymes. We highlight the impact of molecular modeling for mechanistically explaining pathophysiological effects establishing understudied CYPs as promising drug targets.

Antinociceptive Efficacy of the µ-Opioid/Nociceptin Peptide-Based Hybrid KGNOP1 in Inflammatory Pain without Rewarding Effects in Mice: An Experimental Assessment and Molecular Docking.

Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund's adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

Structure-based identification of dual ligands at the A2AR and PDE10A with anti-proliferative effects in lung cancer cell-lines.

Enhanced/prolonged cAMP signalling has been suggested as a suppressor of cancer proliferation. Interestingly, two key modulators that elevate cAMP, the A2A receptor (A2AR) and phosphodiesterase 10A (PDE10A), are differentially co-expressed in various types of non-small lung cancer (NSCLC) cell-lines. Thus, finding dual-target compounds, which are simultaneously agonists at the A2AR whilst also inhibiting PDE10A, could be a novel anti-proliferative approach. Using ligand- and structure-based modelling combined with MD simulations (which identified Val84 displacement as a novel conformational descriptor of A2AR activation), a series of known PDE10A inhibitors were shown to dock to the orthosteric site of the A2AR. Subsequent in-vitro analysis confirmed that these compounds bind to the A2AR and exhibit dual-activity at both the A2AR and PDE10A. Furthermore, many of the compounds exhibited promising anti-proliferative effects upon NSCLC cell-lines, which directly correlated with the expression of both PDE10A and the A2AR. Thus, we propose a structure-based methodology, which has been validated in in-vitro binding and functional assays, and demonstrated a promising therapeutic value.

Exploiting Water Dynamics for Pharmacophore Screening.

Three-dimensional pharmacophore models have been proven extremely valuable in exploring novel chemical space through virtual screening. However, traditional pharmacophore-based approaches need ligand information and rely on static snapshots of highly dynamic systems. In this chapter, we describe PyRod, a novel tool to generate three-dimensional pharmacophore models based on water traces of a molecular dynamics simulation of an apo-protein.The protocol described herein was successfully applied for the discovery of novel drug-like inhibitors of West Nile virus NS2B-NS3 protease. By using this recent example, we highlight the key steps of the generation and validation of PyRod-derived pharmacophore models and their application for virtual screening.

Allosteric coupling and biased agonism in G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are essential cell membrane signaling molecules and represent the most important class of drug targets. Some signaling pathways downstream of a GPCR may be responsible for drug adverse effects, while others mediate therapeutic efficacy. Biased ligands preferentially activate only a subset of all GPCR signaling pathways. They hold great potential to become next-generation GPCR drugs with less side effects due to their potential to exclusively activate desired signaling pathways. However, the molecular basis of biased agonism is poorly understood. GPCR activation occurs through allosteric coupling, the propagation of conformational changes from the extracellular ligand-binding pocket to the intracellular G protein-binding interface. Comparison of GPCR structures in complex with G proteins or ß-arrestin reveals that intracellular transducer coupling results in closure of the ligand-binding pocket trapping the agonist inside its binding site. Allosteric coupling appears to be transducer-specific offering the possibility of harnessing this mechanism for the design of biased ligands. Here, we review the biochemical, pharmacological, structural, and biophysical evidence for allosteric coupling and delineate that biased agonism should be a consequence of preferential allosteric coupling from the ligand-binding pocket to one transducer-binding site. As transducer binding leads to large structural rearrangements in the extracellular ligand-binding pocket, we survey biased ligands with an extended binding mode that interact with extracellular receptor domains. We propose that biased ligands use ligand-specific triggers inside the binding pocket that are relayed through preferential allosteric coupling to a specific transducer, eventually leading to biased signaling.

The Role of Orthosteric Building Blocks of Bitopic Ligands for Muscarinic M1 Receptors.

The muscarinic M1 acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M1 receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M1 receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M1-selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since ß-arrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and ß-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate ß-arrestin recruitment, and the degree of ß-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, ß-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of ß-arrestin recruitment during ligand design for therapeutic usage.

Ligand-Specific Allosteric Coupling Controls G-Protein-Coupled Receptor Signaling.

Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.e., biased) engagement of downstream effectors. However, the structural basis underlying ligand-dependent control of this essential allosteric mechanism is poorly understood. Here, we show that two sets of extended muscarinic acetylcholine receptor M1 agonists, which only differ in linker length, progressively constrain receptor signaling. We demonstrate that stepwise shortening of their chemical linker gradually hampers binding pocket closure, resulting in divergent coupling to distinct G-protein families. Our data provide an experimental strategy for the design of ligands with selective G-protein recognition and reveal a potentially general mechanism of ligand-specific allosteric coupling.

Mechanistic Understanding of Peptide Analogues, DALDA, [Dmt1]DALDA, and KGOP01, Binding to the mu Opioid Receptor.

The mu opioid receptor (MOR) is the primary target for analgesia of endogenous opioid peptides, alkaloids, synthetic small molecules with diverse scaffolds, and peptidomimetics. Peptide-based opioids are viewed as potential analgesics with reduced side effects and have received constant scientific interest over the years. This study focuses on three potent peptide and peptidomimetic MOR agonists, DALDA, [Dmt1]DALDA, and KGOP01, and the prototypical peptide MOR agonist DAMGO. We present the first molecular modeling study and structure-activity relationships aided by in vitro assays and molecular docking of the opioid peptide analogues, in order to gain insight into their mode of binding to the MOR. In vitro binding and functional assays revealed the same rank order with KGOP01 > [Dmt1]DALDA > DAMGO > DALDA for both binding and MOR activation. Using molecular docking at the MOR and three-dimensional interaction pattern analysis, we have rationalized the experimental outcomes and highlighted key amino acid residues responsible for agonist binding to the MOR. The Dmt (2',6'-dimethyl-L-Tyr) moiety of [Dmt1]DALDA and KGOP01 was found to represent the driving force for their high potency and agonist activity at the MOR. These findings contribute to a deeper understanding of MOR function and flexible peptide ligand-MOR interactions, that are of significant relevance for the future design of opioid peptide-based analgesics.

Biological Characterization, Mechanistic Investigation and Structure-Activity Relationships of Chemically Stable TLR2 Antagonists.

Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1-9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1-9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern.

Identification and validation of a novel dual small-molecule TLR2/8 antagonist.

Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.