Ligand Screening Research Articles

Rational drug design targeting g-protein-coupled receptors: a structural biology perspective

G protein-coupled Receptors (G protein-coupled Receptors, GPCRs) play a key role in the transmission of extracellular signals and regulation of many biological processes, which makes these membrane proteins one of the most important classes of targets for pharmacological agents. The significant increase in the number of atomic structures of GPCRs recently has paved the way for Structure Based Drug Design (SBDD). SBDD uses information on the structure of the receptor-ligand complex to search for affinity and selective ligands without the need for high-throughput experimental ligand screening and allows a significant expansion of the chemical ligand search space. In our review we describe the process of GPCR structure obtaining by X-ray diffraction analysis and cryo-electron microscopy (cryo-EM) – an important step in rational drug design targeting GPCRs. Our main goal is to highlight to a wide range of specialists the current aspects and key features of experimental structural biology methods necessary for a detailed understanding of SBDD GPCRs.

Exploring Brazilian Natural Products as Potential Bioactive Compounds against Trypanosoma cruzi by Targeting Squalene Synthase

Introduction: Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi that affects 7 million people worldwide. The current treatment is limited due to safety and efficacy issues. Therefore, the search for new antiparasitic drugs is fundamental. The enzyme squalene synthase (SQS) is an attractive therapeutic target since it participates in the ergosterol biosynthesis pathway. Objective: In the present study, we explored the Brazilian biodiversity to search for potential inhibitors of T. cruzi SQS (TcSQS) using ligand and structure-based virtual screening strategies. Materials and Methods: A virtual screening was performed within the NUBBE database, with more than 2,200 natural products (NP) or semisynthetic derivatives from the Brazilian biodiversity. Molecular docking and ADMET predictions were then performed. Results: A set of 12 NP showed interactions with TcSQS like those observed by known inhibitors and shared literature evidence that supports the predicted activity. Conclusion: Three compounds (flavonoids) showed good ADMET properties as potential inhibitors of TcSQS.

Screening of BindingDB database ligands against EGFR, HER2, Estrogen, Progesterone and NF-[formula omitted]B receptors based on machine learning and molecular docking

Breast cancer, the second most prevalent cancer among women worldwide, necessitates the exploration of novel therapeutic approaches. To target the four subgroups of breast cancer “hormone receptor-positive and HER2-negative, hormone receptor-positive and HER2-positive, hormone receptor-negative and HER2-positive, and hormone receptor-negative and HER2-negative” it is crucial to inhibit specific targets such as EGFR, HER2, ER, NF-κB, and PR.In this study, we evaluated various methods for binary and multiclass classification. Among them, the GA-SVM-SVM:GA-SVM-SVM model was selected with an accuracy of 0.74, an F1-score of 0.73, and an AUC of 0.92 for virtual screening of ligands from the BindingDB database. This model successfully identified 4454, 803, 438, and 378 ligands with over 90% precision in both active/inactive and target prediction for the classes of EGFR+HER2, ER, NF-κB, and PR, respectively, from the BindingDB database. Based on to the selected ligands, we created a dendrogram that categorizes different ligands based on their targets. This dendrogram aims to facilitate the exploration of chemical space for various therapeutic targets.Ligands that surpassed a 90% threshold in the product of activity probability and correct target selection probability were chosen for further investigation using molecular docking. The binding energy range for these ligands against their respective targets was calculated to be between −15 and −5 kcal/mol. Finally, based on general and common rules in medicinal chemistry, we selected 2, 3, 3, and 8 new ligands with high priority for further studies in the EGFR+HER2, ER, NF-κB, and PR classes, respectively.

Synthesis, Urease Inhibition, Molecular Docking, and Optical Analysis of a Symmetrical Schiff Base and Its Selected Metal Complexes.

Designing and developing small organic molecules for use as urease inhibitors is challenging due to the need for ecosystem sustainability and the requirement to prevent health risks related to the human stomach and urinary tract. Moreover, imaging analysis is widely utilized for tracking infections in intracellular and in vivo systems, which requires drug molecules with emissive potential, specifically in the low-energy region. This study comprises the synthesis of a Schiff base ligand and its selected transition metals to evaluate their UV/fluorescence properties, inhibitory activity against urease, and molecular docking. Screening of the symmetrical cage-like ligand and its metal complexes with various eco-friendly transition metals revealed significant urease inhibition potential. The IC50 value of the ligand for urease inhibition was 21.80 ± 1.88 µM, comparable to that of thiourea. Notably, upon coordination with transition metals, the ligand-nickel and ligand-copper complexes exhibited even greater potency than the reference compound, with IC50 values of 11.8 ± 1.14 and 9.31 ± 1.31 µM, respectively. The ligand-cobalt complex exhibited an enzyme inhibitory potential comparable with thiourea, while the zinc and iron complexes demonstrated the least activity, which might be due to weaker interactions with the investigated protein. Meanwhile, all the metal complexes demonstrated a pronounced optical response, which could be utilized for fluorescence-guided targeted drug delivery applications in the future. Molecular docking analysis and IC50 values from in vitro urease inhibition screening showed a trend of increasing activity from compounds 7d to 7c to 7b. Enzyme kinetics studies using the Lineweaver-Burk plot indicated mixed-type inhibition against 7c and non-competitive inhibition against 7d.

Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC’s extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening.

A human lectin array for characterizing host-pathogen interactions

A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal micro-organisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently-labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of micro-organisms.

Identification of Novel Compounds Targeting the Liver X Receptor (LXR): In-silico Studies, Screening, Molecular Docking, and Chemico-pharmacokinetic Analysis

Studies have demonstrated the association between LXR activity dysregulation with many diseases, including atherosclerosis, diabetes and cancer. In recent years, several LXR agonists have surfaced, but none have been approved for human use due to adverse effects or unforeseen reasons. In this study, we first analysed the mRNA and protein expression of LXRs across tissues, network and pathway analysis, and reinterpreted their physiological function and disease association by utilizing multiple biological data repositories, including RNA-seq human protein atlas, DisGeNET, etc. Then, we performed ligand-based virtual screening, chemico-pharmacokinetic analysis, docking and simulation to identify potential new compounds. Our findings of mRNA, protein expression, network and disease enrichment analysis reveal diverse physiological functions of LXRs addressing the possibility of pharmacological manipulation with small molecules would provide therapeutic strategies for disease management. Evaluation of the docking and chemico-pharmacokinetic properties directed to the selection of LXR-623 and AZ876 as promising candidates for LXR-α and LXR-β for further in-silico investigation. Comprehensive screening for new ligands targeting LXRs based on the chemical structures of LXR-623 and AZ876, identified ZINC000005399501 and ZINC000021912941 with the highest binding affinity (−9.8 and −10.7 kcal/mol) for LXRα and LXRβ, respectively. Our results also supported in simulation study, along with favorable chemico-pharmacokinetic features.

Online ligand screening for cytochrome C from herbal medicines through “four-in-one” measurement

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC–energy-resolved-affinity MS (PCI-LC–ER-AMS) to achieve “four-in-one” monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC–ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through “four-in-one” measurement.

Exploring molecular interactions and ADMET profiles of novel MAO-B inhibitors: toward effective therapeutic strategies for neurodegenerative disorders

BackgroundNeurodegenerative disorders (NDs), primarily affecting the elderly, are marked by complex pathophysiological processes and are projected to become the second leading cause of death. Parkinson’s disease (PD), one of the most common NDs, is characterized by motor impairments due to reduced dopamine levels in the substantia nigra (SN), a crucial midbrain region involved in motor control and reward mechanisms. PD also impacts cognitive functions, potentially leading to depression and sleep disturbances. Recent research highlights the importance of MAO-B inhibitors in PD management, as these enzymes play a critical role in regulating neurotransmitter levels by catalyzing the oxidative deamination of intracellular amines and monoamine neurotransmitters.ResultComputational virtual screening of several quinoline-based ligands against the target protein MAO-B (PDB ID: 1OJA) was performed using molecular docking simulation and ADMET studies to identify promising inhibitors for neurodegenerative disease treatment. The most active hit, Compound PA001, exhibited a MolDock score of − 207.76 kcal/mol. Subsequent investigation of 6-methoxy-2-(4-phenylpiperazin-1-yl)quinoline (Compound PA001) using molecular dynamics (MD) simulations with GROMACS revealed potent inhibition and significant interactions at key active site residues. MD simulations confirmed the stability of the Compound PA001-MAO-B complex under physiological conditions. Additionally, ADMET analysis demonstrated that Compound PA001 possesses favorable drug-like properties, including absorption, distribution, metabolism, excretion, and toxicity profiles. These findings underscore 6-methoxy-2-(4-phenylpiperazin-1-yl)quinoline (Compound PA001) as a promising candidate for developing new MAO-B inhibitors to treat neurodegenerative diseases.ConclusionThe research highlighted 6-methoxy-2-(4-phenylpiperazin-1-yl)quinoline (Compound PA001) as a promising MAO-B inhibitor, exhibiting strong binding affinity, stability, and desirable drug-like characteristics for the treatment of neurodegenerative diseases. Among the top ten molecules, Compound PA001 was selected for molecular dynamics (MD) simulation using GROMACS. The compound showed potent inhibition, significant interactions with key active site residues, and stable complex formation under physiological conditions. ADMET analysis further confirmed its favorable pharmacokinetic profile.

Ligand and structure-based virtual screening approaches in drug discovery: minireview.

The compilation of ligand and structure-based molecular modeling methods has become an important practice in virtual screening applied to drug discovery. This systematic review addresses and ranks various virtual screening strategies to drive the selection of the optimal method for studies that have as their starting point a multi-ligand investigation and investigation based on the protein structure of a therapeutic target. This study shows examples of applications and an evaluation based on the objective and problematic of a series of virtual screening studies present in the ScienceDirect® database. The results showed that the molecular docking technique is widely used in scientific production, indicating that approaches that use protein structure as a starting point are the most promising strategy for drug discovery that relies on virtual screening-based research.

Selective Clearance of Circulating Histones Based on Dodecapeptide-Grafted Copolymer Material for Sepsis Blood Purification.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Research indicates that circulating histones, as pathogenic factors, may represent a therapeutic target for sepsis. However, effectively clearing circulating histones poses a challenge due to their structural similarity to normal blood proteins, their low abundance in the bloodstream, and serious interference from other blood biomacromolecules. Here we design a dodecapeptide-based functional polymer that can selectively adsorb circulating histones from the blood. The peptide, named P1 (HNHHQLALVESY), was discovered through phage display screening and demonstrated a strong affinity for circulating histones while exhibiting negligible affinities for common proteins in the blood, such as human serum albumin (HSA), immunoglobulin G (IgG), and transferrin (TRF). Furthermore, the P1 peptide was incorporated into a functional polymer design, poly(PEGMA-co-P1), which was immobilized onto a silica gel surface through reversible addition-fragmentation chain transfer polymerization. The resulting material was characterized using solid nuclear magnetic resonance, thermogravimetric analysis, and X-ray photoelectron spectroscopy. This material demonstrated the ability to selectively and efficiently capture circulating histones from both model solutions and whole blood samples while also exhibiting satisfactory blood compatibility, good antifouling properties, and resistance to interference. Satisfactory binding affinity and efficient capture capacity toward histone were also observed for the other screened peptide P2 (QMSMDLFGSNFV)-grafted polymer, validating phage display as a reliable ligand screening strategy. These findings present an approach for the specific clearance of circulating histones and hold promise for future clinical applications in blood purification toward sepsis.

Structure-Based Drug Design with a Deep Hierarchical Generative Model.

Recently, the remarkable growth of available crystal structure data and libraries of commercially available or readily synthesizable molecules have unlocked previously inaccessible regions of chemical space for drug development. Paired with improvements in virtual ligand screening methods, these expanded libraries are having a notable impact on early drug design efforts. Yet screening-based methods still face scalability limits, due to computational constraints and the sheer scale of drug-like space. Machine learning approaches are overcoming these limitations by learning the fundamental intra- and intermolecular relationships in drug-target systems from existing data. Here, we introduce DrugHIVE, a deep hierarchical variational autoencoder that outperforms state-of-the-art autoregressive and diffusion-based methods in both speed and performance on common generative benchmarks. DrugHIVE's hierarchical design enables improved control over molecular generation. Its capabilities include dramatically increasing virtual screening efficiency and accelerating a wide range of common drug design tasks, including de novo generation, molecular optimization, scaffold hopping, linker design, and high-throughput pattern replacement. Our highly scalable method can even be applied to receptors with high-confidence AlphaFold-predicted structures, extending the ability to generate high-quality drug-like molecules to a majority of the unsolved human proteome.

Design, synthesis, anti-tumor activity and mechanism of novel PROTACs as degraders of PD-L1 and inhibitors of PD-1/PD-L1 interaction

Currently, antibody drugs targeting programmed cell death ligand 1 (PD-L1) have achieved promising results in cancer treatment, while the development of small-molecule drugs lags behind. In this study, we designed and synthesized a series of PD-L1-degrading agents based on the PROTAC design principle, utilizing the PD-L1 inhibitor A56. Through systematic screening of ligands and linkers and investigating the structure–activity relationship of the degraders, we identified two highly active compounds, 9i and 9j. These compounds enhance levels of CD4+, CD8+, granzyme B, and perforin, demonstrating significant in vivo antitumor effects with a tumor growth inhibition (TGI) of up to 57.35 %. Both compounds facilitate the internalization of PD-L1 from the cell surface and promote its degradation through proteasomal and lysosomal pathways, while also maintaining inhibition of the PD-1/PD-L1 interaction. In summary, our findings provide a novel strategy and mechanism for developing biphenyl-based PROTAC antitumor drugs targeting and degrading PD-L1.

Human AKR1C3 binds agonists of GPR84 and participates in an expanded polyamine pathway.

Altered human aldo-keto reductase family 1 member C3 (AKR1C3) expression has been associated with poor prognosis in diverse cancers, ferroptosis resistance, and metabolic diseases. Despite its clinical significance, the endogenous biochemical roles of AKR1C3 remain incompletely defined. Using untargeted metabolomics, we identified a major transformation mediated by AKR1C3, in which a spermine oxidation product "sperminal" is reduced to "sperminol." Sperminal causes DNA damage and activates the DNA double-strand break response, whereas sperminol induces autophagy invitro. AKR1C3 also pulls down acyl-pyrones and pyrone-211 inhibits AKR1C3 activity. Through G protein-coupled receptor ligand screening, we determined that pyrone-211 is also a potent agonist of the semi-orphan receptor GPR84. Strikingly, mammalian fatty acid synthase produces acyl-pyrones invitro, and this production is modulated by NADPH. Taken together, our studies support a regulatory role of AKR1C3 in an expanded polyamine pathway and a model linking fatty acid synthesis and NADPH levels to GPR84 signaling.

Palladium‐Catalyzed Asymmetric Allylic Substitutions Achieved by C–N Axially Chiral N‐Arylpyrrole Derived Monophosphine Ligands

Monophosphine ligands based on C–N axially chiral N‐Arylpyrrole backbones are prepared starting from amino acids and evaluated in Pd‐catalyzed asymmetric allylic substitutions. 20.7‐ 99.9% ees are achieved in the reactions of rac‐1,3‐diphenylallyl acetates with O‐, N‐ and C‐nucleophiles. Ligand screening revealed that the steric hindrance from 3‐ or 4‐ position of the pyrrole is crucial for the enantioselectivity of the reaction. The synthetic utilization of the products was demonstrated by the transformation of one product by a gold‐catalyzed oxidative rearrangement reaction.

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.