Ligand Screening Research Articles

Jellyfish Venom Peptides Targeting Human Potassium Channels Identified through Ligand Screening: Morphometric and Molecular Identification of the Species and Antibiotic Potential.

The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.

MrgX2-targeted ligand screening from Artemisia capillaris Thunb. extract and receptor-ligand interaction analysis based on MrgX2-HALO-tag/CMC

Artemisia capillaris Thunb. (A. capillaris) is a well-known traditional Chinese herbal medicine with a wide range of pharmacological effects, such as soothing the liver and gallbladder, heat clearance, and detoxifying. Hence, its extract is commonly added to various traditional Chinese medicine formulas. Traditional Chinese medicine injection (TCMI) is a mature pharmaceutical dosage form developed using TCM theory combined with modern science and technology. Notably, allergic reactions, especially pseudo‑allergic reactions (PARs), greatly limited the use of these injections. Therefore, screening pseudo‑allergic components in A. capillaris extract is clinically significant. In the present study, we proposed a two-dimensional screening and identification system based on mas-related G protein-coupled receptor X2-HALO-tag/cell membrane chromatography (MrgX2-HALO-tag/CMC) high performance liquid chromatography mass spectrometry (HPLC-MS); seven potential active components were screened from 75 % ethanol extract of A. capillaris: NCA, CA, CCA, 1,3-diCQA, ICA-B, ICA-A, and ICA-C. The receptor-ligand interactions between these seven compounds and MrgX2 protein were analyzed using frontal analysis and molecular docking technology. Furthermore, a mast cell degranulation-related assay was used to assess the pseudo‑allergic activity of these compounds. The screened compounds can serve as ligands of MrgX2, and this study provides a research basis for pseudo‑allergic reactions caused by TCMIs containing A. capillaris.

RhoA-mediated G12-G13 signaling maintains muscle stem cell quiescence and prevents stem cell loss

Multiple processes control quiescence of muscle stem cells (MuSCs), which is instrumental to guarantee long-term replenishment of the stem cell pool. Here, we describe that the G-proteins G12-G13 integrate signals from different G-protein-coupled receptors (GPCRs) to control MuSC quiescence via activation of RhoA. Comprehensive screening of GPCR ligands identified two MuSC-niche-derived factors, endothelin-3 (ET-3) and neurotensin (NT), which activate G12-G13 signaling in MuSCs. Stimulation with ET-3 or NT prevented MuSC activation, whereas pharmacological inhibition of ET-3 or NT attenuated MuSC quiescence. Inactivation of Gna12-Gna13 or Rhoa but not of Gnaq-Gna11 completely abrogated MuSC quiescence, which depleted the MuSC pool and was associated with accelerated sarcopenia during aging. Expression of constitutively active RhoA prevented exit from quiescence in Gna12-Gna13 mutant MuSCs, inhibiting cell cycle entry and differentiation via Rock and formins without affecting Rac1-dependent MuSC projections, a hallmark of quiescent MuSCs. The study uncovers a critical role of G12-G13 and RhoA signaling for active regulation of MuSC quiescence.

A Benchmark Test of High-Throughput Atomistic Modeling for Octa-Acid Host–Guest Complexes

Years of massive applications of high-throughput atomistic modeling tools such as molecular docking and end-point free energy calculations in the drug industry and academic exploration have made them indispensable parts of hierarchical screening. While the similarities between host–guest and protein–ligand complexes lead to the direct extension of techniques for protein–ligand screening to host–guest systems, the practical performance of these hit identification tools remains unclear in host-–-guest binding. Recent reports on specific host–guest complexes suggest that the experience on the accuracy ladder accumulated from protein–ligand cases could be invalid in host–guest complexes, which makes it an urgent need to perform a systematic benchmark to secure solid numerical supports and guidance of practical setups. Concerning molecular docking, there still lacks a comprehensive benchmark considering popular docking programs. As for end-point reranking, quantitative and rigorous free energy estimation via end-point formulism requires establishing statistically meaningful measurements of uncertainties due to finite sampling, which is neglected or underestimated by a significant portion in almost all main-stream applications. Further, a face-to-face comparison between different screening tools is required for the design of a hierarchical workflow. To fill the above-mentioned critical gaps, in this work, using a dataset containing tens of host–guest complexes involving basket-like macromolecular hosts from the octa acid family, we extensively benchmark seven academic docking protocols and perform post-docking end-point rescoring with twenty protocols. The resulting comprehensive benchmark provides conclusive pictures of the practical value of docking and end-point screening in OA host–guest binding.

Screening of inhibitors on successful covalent tyrosinase coupling with help from SpyBank.

Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag-TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag-TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1H NMR and 13C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems.

The Chameleon Strategy-A Recipe for Effective Ligand Screening for Viral Targets Based on Four Novel Structure-Binding Strength Indices.

The RNA viruses SARS-CoV, SARS-CoV-2 and MERS-CoV encode the non-structural Nsp16 (2'-O-methyltransferase) that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the first ribonucleotide in mRNA. Recently, it has been found that breaking the bond between Nsp16 and SAM substrate results in the cessation of mRNA virus replication. To date, only a limited number of such inhibitors have been identified, which can be attributed to a lack of an effective "recipe". The aim of our study was to propose and verify a rapid and effective screening protocol dedicated to such purposes. We proposed four new indices describing structure-binding strength (structure-binding affinity, structure-hydrogen bonding, structure-steric and structure-protein-ligand indices) were then applied and shown to be extremely helpful in determining the degree of increase or decrease in binding affinity in response to a relatively small change in the ligand structure. After initial pre-selection, based on similarity to SAM, we limited the study to 967 compounds, so-called molecular chameleons. They were then docked in the Nsp16 protein pocket, and 10 candidate ligands were selected using the novel structure-binding affinity index. Subsequently the selected 10 candidate ligands and 8 known inhibitors and were docked to Nsp16 pockets from SARS-CoV-2, MERS-CoV and SARS-CoV. Based on the four new indices, the best ligands were selected and a new one was designed by tuning them. Finally, ADMET profiling and molecular dynamics simulations were performed for the best ligands. The new structure-binding strength indices can be successfully applied not only to screen and tune ligands, but also to determine the effectiveness of the ligand in response to changes in the target viral entity, which is particularly useful for assessing drug effectiveness in the case of alterations in viral proteins. The developed approach, the so-called chameleon strategy, has the capacity to introduce a novel universal paradigm to the field of drugs design, including RNA antivirals.

Enantioselective Synthesis of Allylic Sulfones via Rhodium-Catalyzed Direct Hydrosulfonylation of Allenes and Alkynes.

A highly regio- and enantioselective hydrosulfonylation using commercially available sodium sulfinates is reported, providing the first direct asymmetric rhodium-catalyzed hydrosulfonylation of allenes/alkynes to synthesize chiral allylic sulfones. Ligand screening studies demonstrated the indispensable role of the C1-symmetric P,N-ligand (Rax,S,S)-StackPhim for achieving both high regioselecitivity (>20:1) and enantioselectivity (up to 97% ee). Notably, the operationally simple method and mild conditions allow for the rapid preparation of chiral allylic sulfones with a wide scope of functional groups. Moreover, the use of sodium tert-butyldimethylsilyloxymethanesulfinate enables the collective synthesis of various chiral sulfone derivatives after simple transformations of the protected hydroxymethyl product.

Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

An NMR Toolkit to Probe Amyloid Oligomer Inhibition in Neurodegenerative Diseases: From Ligand Screening to Dissecting Binding Topology and Mechanisms of Action.

The aggregation of amyloid peptides and proteins into toxic oligomers is a hallmark of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Machado-Joseph's disease, and transmissible spongiform encephalopathies. Inhibition of amyloid oligomers formation and interactions with biological counterparts, as well as the triggering of non-toxic amorphous aggregates, are strategies towards preventive interventions against these pathologies. NMR spectroscopy addresses the need for structural characterization of amyloid proteins and their aggregates, their binding to inhibitors, and rapid screening of compound libraries for ligand identification. Here we briefly discuss the solution experiments constituting the NMR spectroscopist's toolkit and provide examples of their application.

Erratum to: Rational Design of Drugs Targeting G-Protein-Coupled Receptors: Ligand Search and Screening.

Erratum to: Rational Design of Drugs Targeting G-Protein-Coupled Receptors: Ligand Search and Screening.

Investigation of the Affinity of Aptamers for Bacteria by Surface Plasmon Resonance Imaging Using Nanosomes.

The cell-SELEX method enables efficient selection of aptamers that bind whole bacterial cells. However, after selection, it is difficult to determine their binding affinities using common screening methods because of the large size of the bacteria. Here we propose a simple surface plasmon resonance imaging method (SPRi) for aptamer characterization using bacterial membrane vesicles, called nanosomes, instead of whole cells. Nanosomes were obtained from membrane fragments after mechanical cell disruption in order to preserve the external surface epitopes of the bacterium used for their production. The study was conducted on Bacillus cereus (B. cereus), a Gram-positive bacterium commonly found in soil, rice, vegetables, and dairy products. Four aptamers and one negative control were initially grafted onto a biochip. The binding of B. cereus cells and nanosomes to immobilized aptamers was then compared. The use of nanosomes instead of cells provided a 30-fold amplification of the SPRi signal, thus allowing the selection of aptamers with higher affinities. Aptamer SP15 was found to be the most sensitive and selective for B. cereus ATCC14579 nanosomes. It was then truncated into three new sequences (SP15M, SP15S1, and SP15S2) to reduce its size while preserving the binding site. Fitting the results of the SPRi signal for B. cereus nanosomes showed a similar trend for SP15 and SP15M, and a slightly higher apparent association rate constant kon for SP15S2, which is the truncation with a high probability of a G-quadruplex structure. These observations were confirmed on nanosomes from B. cereus ATCC14579 grown in milk and from the clinical strain B. cereus J066. The developed method was validated using fluorescence microscopy on whole B. cereus cells and the SP15M aptamer labeled with a rhodamine. This study showed that nanosomes can successfully mimic the bacterial membrane with great potential for facilitating the screening of specific ligands for bacteria.

Utility of the Morgan Fingerprint in Structure-Based Virtual Ligand Screening.

In modern drug discovery, virtual ligand screening (VLS) is frequently applied to identify possible hits before experimental testing and refinement due to its cost-effective nature for large compound libraries. For decades, efforts have been devoted to developing VLS methods with high accuracy. These include the state-of-the-art FINDSITE suite of approaches FINDSITEcomb2.0, FRAGSITE, and FRAGSITE2 and the meta version FRAGSITEcomb that were developed in our lab. These methods combine ligand homology modeling (LHM), traditional ligand similarity methods, and more recently machine learning approaches to rank ligands and have proven to be superior to most recent deep learning and large language model-based approaches. Here, we describe further improvements to our previous best methods by combining the Morgan fingerprint (MF) with the originally used PubChem fingerprint and FP2 fingerprint. We then benchmarked FINDSITEcomb2.0M, FRAGSITEM, FRAGSITE2M, and the composite meta-approach FRAGSITEcombM. On the 102 target DUD-E set, the 1% enrichment factor (EF1%) and area under the precision-recall curve (AUPR) of FRAGSITEcomb increased from 42.0/0.59 to 47.6/0.72. This 0.72 AUPR is significantly better than that of the state-of-the-art deep learning-based method DenseFS's AUPR of 0.443. An independent test on the 81 targets DEKOIS2.0 set shows that EF1%/AUPR increases from 18.3/0.520 to 23.1/0.683. An ablation investigation shows that the MF contributes to most of the improvement of all four approaches. Thus, the MF is a useful addition to structure-based VLS.

Exploring solute binding proteins in Pseudomonas aeruginosa that bind to γ-aminobutyrate and 5-aminovalerate and their role in activating sensor kinases.

The standard method of receptor activation involves the binding of signals or signal-loaded solute binding proteins (SBPs) to sensor domains. Many sensor histidine kinases (SHKs), which are activated by SBP binding, are encoded adjacent to their corresponding sbp gene. We examined three SBPs of Pseudomonas aeruginosa PAO1, encoded near the genes for the AgtS (PA0600) and AruS (PA4982) SHKs, to determine how common this arrangement is. Ligand screening and microcalorimetric studies revealed that the SBPs PA0602 and PA4985 preferentially bind to GABA (KD = 2.3 and 0.58 μM, respectively), followed by 5-aminovalerate (KD = 30 and 1.6 μM, respectively) and ethanoldiamine (KD = 2.3 and 0.58 μM, respectively). In contrast, AgtB (PA0604) exclusively recognizes 5-aminovaleric acid (KD = 2.9 μM). However, microcalorimetric titrations did not show any binding between the AgtS sensor domain and AgtB or PA0602, regardless of the presence of ligands. Similarly, bacterial two-hybrid assays did not demonstrate an interaction between PA4985 and the AruS sensor domain. Therefore, sbp and shk genes located nearby are not always functionally linked. We previously identified PA0222 as a GABA-specific SBP. The presence of three SBPs for GABA may be linked to GABA's role as a trigger for P. aeruginosa virulence.

Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.

Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.

Exploring the bonding and extraction separation effects of N donor ligands with different skeletons on Am(III) and Eu(III)

Designing ligands for effective separation of actinide(III)/lanthanide(III) is one of the key issues in the treatment of spent nuclear fuel. However, due to the chemical similarity of trivalent actinides and lanthanides, their separation faces a great challenge in the reprocessing of spent nuclear fuel. Nitrogen donor extractants are considered as promising reagents for separation of trivalent actinides and lanthanides. To the best of our knowledge, the pre-organized structure of the ligand has a strong influence on the extraction separation capacity. In this work, we designed four N-donor ligands (L1 and L2 with pyridine and bipyridine skeletons, respectively, and L3 and L4 both with phenanthroline skeletons) with different pre-organized structures and investigated their selective extraction for Am(III) and Eu(III) by using scalar relativistic density functional theory (DFT). The absolute minimum ESP values of these ligands and the higher HOMO level suggest that the ligand L2 may have a stronger affinity for Am(III)/Eu(III) ions than other ligands. The three average bond lengths of each complex indicate that the Am(III) and Eu(III) ions have larger bond lengths with N2 atoms than with N1 atoms. Various theoretical approaches have been used to evaluate the properties of the four ligands as well as the coordination structures, bonding properties and thermodynamic properties of the complexes of the four ligands with Am(III) and Eu(III). The WBIs, QTAIM, NBO, DOS and EDA analysis indicate that the metal–ligand bonds are mainly ionic in character, the Am-N bond has more covalency than the Eu-N bond because the Am 5f orbitals are more involved in bonding with ligand than Eu 4f orbitals. According to the extended transition state and natural orbital chemical valence theory (ETS-NOCV) analysis, electrons are obviously transferred from the lone pair of the ligand N atom to the hybrid s, d and f orbitals of the metal ion. The thermodynamic results show that the four ligands can coordinate well with the Am and Eu ions, and also have suitable separation capacity for Am and Eu. Among the four ligands, the L2 and L3 ligands have the strongest coordination ability for metals and the best extraction separation effect for Am and Eu. It can be seen that the bridging skeleton of the ligand and the number of N atoms on the five-membered ring and also different organic solvents have obvious effects on the extraction and separation capacity of the ligand. This study provides a theoretical basis for the efficient separation of Am(III)/Eu(III) by adjusting the preorganization level of ligands, and provides a theoretical basis for the design and screening of ligands by preorganization.

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors

BackgroundPeroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. ResultsIn this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SignificanceA new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

Signaling of GPR17 in Hepatic and Renal Tissues

G protein-coupled receptors (GPCRs) are the largest class of proteins in the human body and are considered to be excellent targets for drug discovery. However, the vast majority of GPCRs are understudied. Using a large-scale TaqMan array screen on both male and female C57BL6 murine livers, we identified GPR17 as one of the most highly expressed orphan receptors within the liver (ΔCt = 19.9, normalized to GAPDH). GPR17 expression in the liver was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR) and was also identified in murine whole kidney from both males and females indicating that GPR17 RNA expression is not sex-dependent. While GPR17 has known roles in oligodendrocyte maturation and myelination in the central nervous system, its functions within the liver and kidney are undetermined. To investigate its physiological functions, we started by uncovering its localization within the liver and kidney and identifying its ligand profile. A series of RNAScope in-situ hybridization assays were performed to localize GPR17 within hepatic tissue. Gpr17 mRNA was found within hepatocytes indicating that its highest expression is in the parenchymal cells of the liver. To identify possible activators for GPR17, we cloned the coding region into both flag-tagged PME18s and the Presto-Tango vectors. Live-cell surface labeling of HEK293T expressing the GPR17 constructs confirmed robust surface expression on the plasma membrane facilitating ligand screening. GPR17 downstream signaling has yet to be studied. Thus, we used the PRESTO-TANGO assay, an exogenous chemical ligand screening assay that relies on a luminescence readout to indicate -arrestin recruitment and receptor activation. A large, non-biased chemical screen of over 500 physiological compounds was tested for activation of GPR17. To date, we identified seven exogenous activators of GPR17: 3-[methyl(1,2,3,4- tetrahydronaphthalen-2-yl)amino]pyrazine-2-carbonitrile, 4-(4-fluorophenyl)-5-methyl-N-(2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)thiophene-2-carboxamide,({1-[(2-chlorophenyl)methyl]-3,5-dimethyl-1H-pyrazol-4-yl}methyl)(methyl)[(1-methyl-1H-imidazol-2-yl)methyl]amine,N,N-dimethyl-6-[(5-phenyl-1,3-oxazol-2-yl)methyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-4-amine,4-phenyl-1-[6-(pyrrolidin-1-yl)-9H-purin-9-yl]butan-2-ol, 2-(3-fluorophenyl)-3-methyl-1-{[1,2,4]triazolo[4,3-a]pyridine-6-carbonyl}pyrrolidine, and 2,6,6-trimethyl-N-({2-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrimidin-6-yl}methyl)-4,5,6,7-tetrahydro-1-benzofuran-4-amine. Ongoing studies are aimed at narrowing down the in vivo activators of GPR17 to ultimately shed light on the physiological functions that this receptor plays in the kidney and liver. NIH/NCATS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rational Design of Drugs Targeting G-Protein-Coupled Receptors: Ligand Search and Screening.

G protein-coupled receptors (GPCRs) are transmembrane proteins that participate in many physiological processes and represent major pharmacological targets. Recent advances in structural biology of GPCRs have enabled the development of drugs based on the receptor structure (structure-based drug design, SBDD). SBDD utilizes information about the receptor-ligand complex to search for suitable compounds, thus expanding the chemical space of possible receptor ligands without the need for experimental screening. The review describes the use of structure-based virtual screening (SBVS) for GPCR ligands and approaches for the functional testing of potential drug compounds, as well as discusses recent advances and successful examples in the application of SBDD for the identification of GPCR ligands.