Identification of Potential Drug Targets of Calix[4]arene by Reverse Docking.

Calixarenes are displaying great potential for the development of new drug delivery systems, diagnostic imaging, biosensing devices and inhibitors of biological processes. In particular, calixarene derivatives are able to interact with many different enzymes and function as inhibitors. By screening of the potential drug target database (PDTD) with a reverse docking procedure, we identify and discuss a selection of 100 proteins that interact strongly with calix[4]arene. We also discover that leucine (23.5 %), isoleucine (11.3 %), phenylalanines (11.3 %) and valine (9.5 %) are the most frequent binding residues followed by hydrophobic cysteines and methionines and aromatic histidines, tyrosines and tryptophanes. Top binders are peroxisome proliferator-activated receptors that already are targeted by commercial drugs, demonstrating the practical interest in calix[4]arene. Nuclear receptors, potassium channel, several carrier proteins, a variety of cancer-related proteins and viral proteins are prominent in the list. It is concluded that calix[4]arene, which is characterized by facile access, well-defined conformational characteristics, and ease of functionalization at both the lower and higher rims, could be a potential lead compound for the development of enzyme inhibitors and theranostic platforms.

Identification and Characterization of Evolutionary Conserved Muskmelon Non-coding miRNAs and Their Target Proteins.

MicroRNAs (miRNAs) are short, endogenously encoded small RNAs, 18-26 nucleotides in length, which can posttranscriptionally regulate gene expression through translation inhibition or endonucleolytic cleavage. The muskmelon is one of the most widely cultivated fruits in the Cucurbitaceae family. Despite its significance, only 120 miRNAs from different families have been reported in muskmelon. In this study, we aimed to expand this knowledge base by predicting 40 new miRNAs in muskmelon using a spectrum of genomic-based tools. Precursor and mature sequences were obtained from microRNA registry database as reference and analyzed via the basic local alignment search tool (Blastn) for ESTs identification. After removing the non-coding sequences, the remaining candidate sequences were analyzed using MFOLD to generate secondary structures for the newly predicted miRNAs. Additionally, the predicted muskmelon miRNAs were validated using a set of five randomly chosen primers and RT-PCR. Through gene ontology (GO) analysis, we identified 89 targets associated with newly predicted muskmelon miRNAs. Transcription factor-coding genes play a crucial role in plant growth and development. Additionally, the miR4249 has been found to have the same targets in muskmelon that have been linked to cell signaling and transcription factors. The identified targets are integral for diverse biological processes including plant growth, development, metabolism, aging, disease resistance, and resistance to environmental stresses, such as salt, cold, and oxidative stress. As a result, the outcomes of this study demonstrate that this mechanism not only contributes to the production of a higher quality crop but also enhances overall production.

Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules.

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification.

Novel Potential Janus Kinase Inhibitors with Therapeutic Prospects in Rheumatoid Arthritis Addressed by In Silico Studies.

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder with an inflammatory condition targeting the joints that affects millions of patients worldwide. Several unmet needs still need to be addressed despite recent improvements in the management of RA. Although current RA therapies can diminish inflammation and alleviate symptoms, many patients remain unresponsive or experience flare-ups of their ailment. The present study aims to address these unmet needs through in silico research, with a focus on the identification of novel, potentially active molecules. Therefore, a molecular docking analysis has been conducted using AutoDockTools 1.5.7 on Janus kinase (JAK) inhibitors that are either approved for RA or in advanced phases of research. The binding affinities of these small molecules against JAK1, JAK2, and JAK3, which are target proteins implicated in the pathophysiology of RA, have been assessed. Subsequent to identifying the ligands with the highest affinity for these target proteins, a ligand-based virtual screening was performed utilizing SwissSimilarity, starting with the chemical structures of the previously identified small molecules. ZINC252492504 had the highest binding affinity (-9.0 kcal/mol) for JAK1, followed by ZINC72147089 (-8.6 kcal/mol) for JAK2, and ZINC72135158 (-8.6 kcal/mol) for JAK3. Using SwissADME, an in silico pharmacokinetic evaluation showed that oral administration of the three small molecules may be feasible. Based on the preliminary results of the present study, additional extensive research is required for the most promising candidates to be conducted so their efficacy and safety profiles can be thoroughly characterized, and they can become medium- and long-term pharmacotherapeutic solutions for the treatment of RA.

LncRNAs in the Regulation of Genes and Signaling Pathways through miRNA-Mediated and Other Mechanisms in Clear Cell Renal Cell Carcinoma.

The fundamental novelty in the pathogenesis of renal cell carcinoma (RCC) was discovered as a result of the recent identification of the role of long non-coding RNAs (lncRNAs). Here, we discuss several mechanisms for the dysregulation of the expression of protein-coding genes initiated by lncRNAs in the most common and aggressive type of kidney cancer-clear cell RCC (ccRCC). A model of competitive endogenous RNA (ceRNA) is considered, in which lncRNA acts on genes through the lncRNA/miRNA/mRNA axis. For the most studied oncogenic lncRNAs, such as HOTAIR, MALAT1, and TUG1, several regulatory axes were identified in ccRCC, demonstrating a number of sites for various miRNAs. Interestingly, the LINC00973/miR-7109/Siglec-15 axis represents a novel agent that can suppress the immune response in patients with ccRCC, serving as a valuable target in addition to the PD1/PD-L1 pathway. Other mechanisms of action of lncRNAs in ccRCC, involving direct binding with proteins, mRNAs, and genes/DNA, are also considered. Our review briefly highlights methods by which various mechanisms of action of lncRNAs were verified. We pay special attention to protein targets and signaling pathways with which lncRNAs are associated in ccRCC. Thus, these new data on the different mechanisms of lncRNA functioning provide a novel basis for understanding the pathogenesis of ccRCC and the identification of new prognostic markers and targets for therapy.

Techniques and Strategies for Potential Protein Target Discovery and Active Pharmaceutical Molecule Screening in a Pandemic.

Viruses remain a major challenge in the fierce fight against diseases. There have been many pandemics caused by various viruses throughout the world over the years. Recently, the global outbreak of COVID-19 has had a catastrophic impact on human health and the world economy. Antiviral drug treatment has become another essential means to overcome pandemics in addition to vaccine development. How to quickly find effective drugs that can control the development of a pandemic is a hot issue that still needs to be resolved in medical research today. To accelerate the development of drugs, it is necessary to target the key target proteins in the development of the pandemic, screen active molecules, and develop reliable methods for the identification and characterization of target proteins based on the active ingredients of drugs. This article discusses key target proteins and their biological mechanisms in the progression of COVID-19 and other major epidemics. We propose a model based on these foundations, which includes identifying potential core targets, screening potential active molecules of core targets, and verifying active molecules. This article summarizes the related innovative technologies and methods. We hope to provide a reference for the screening of drugs related to pandemics and the development of new drugs.

Opening up connectivity between documents, structures and bioactivity.

Bioscientists reading papers or patents strive to discern the key relationships reported within a document "D" where a bioactivity "A" with a quantitative result "R" (e.g., an IC50) is reported for chemical structure "C" that modulates (e.g., inhibits) a protein target "P". A useful shorthand for this connectivity thus becomes DARCP. The problem at the core of this article is that the community has spent millions effectively burying these relationships in PDFs over many decades but must now spend millions more trying to get them back out. The key imperative for this is to increase the flow into structured open databases. The positive impacts will include expanded data mining opportunities for drug discovery and chemical biology. Over the last decade commercial sources have manually extracted DARCP from ≈300,000 documents encompassing ≈7 million compounds interacting with ≈10,000 targets. Over a similar time, the Guide to Pharmacology, BindingDB and ChEMBL have carried out analogues DARCP extractions. Although their expert-curated numbers are lower (i.e., ≈2 million compounds against ≈3700 human proteins), these open sources have the great advantage of being merged within PubChem. Parallel efforts have focused on the extraction of document-to-compound (D-C-only) connectivity. In the absence of molecular mechanism of action (mmoa) annotation, this is of less value but can be automatically extracted. This has been significantly accomplished for patents, (e.g., by IBM, SureChEMBL and WIPO) for over 30 million compounds in PubChem. These have recently been joined by 1.4 million D-C submissions from three major chemistry publishers. In addition, both the European and US PubMed Central portals now add chemistry look-ups from abstracts and full-text papers. However, the fully automated extraction of DARCLP has not yet been achieved. This stands in contrast to the ability of biocurators to discern these relationships in minutes. Unfortunately, no journals have yet instigated a flow of author-specified DARCP directly into open databases. Progress may come from trends such as open science, open access (OA), findable, accessible, interoperable and reusable (FAIR), resource description framework (RDF) and WikiData. However, we will need to await the technical applicability in respect to DARCP capture to see if this opens up connectivity.

Prediction of Protein-Ligand Interaction Based on the Positional Similarity Scores Derived from Amino Acid Sequences.

The affinity of different drug-like ligands to multiple protein targets reflects general chemical-biological interactions. Computational methods estimating such interactions analyze the available information about the structure of the targets, ligands, or both. Prediction of protein-ligand interactions based on pairwise sequence alignment provides reasonable accuracy if the ligands' specificity well coincides with the phylogenic taxonomy of the proteins. Methods using multiple alignment require an accurate match of functionally significant residues. Such conditions may not be met in the case of diverged protein families. To overcome these limitations, we propose an approach based on the analysis of local sequence similarity within the set of analyzed proteins. The positional scores, calculated by sequence fragment comparisons, are used as input data for the Bayesian classifier. Our approach provides a prediction accuracy comparable or exceeding those of other methods. It was demonstrated on the popular Gold Standard test sets, presenting different sequence heterogeneity and varying from the group, including different protein families to the more specific groups. A reasonable prediction accuracy was also found for protein kinases, displaying weak relationships between sequence phylogeny and inhibitor specificity. Thus, our method can be applied to the broad area of protein-ligand interactions.

In Silico and In Vitro Anti-Helicobacter Pylori Effects of Combinations of Phytochemicals and Antibiotics.

Helicobacter pylori infection is a WHO class 1 carcinogenic factor of gastric adenocarcinoma. In the past decades, many studies have demonstrated the increasing trend of antibiotic resistance and pointed out the necessity of new effective treatment. This study was aimed at identifying phytochemicals that can inhibit H. pylori and possibly serve as adjuvant treatments. Here, in silico molecular docking and drug-like properties analyses were performed to identify potential inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. These three enzymes are targets of the treatment of H. pylori. Susceptibility and synergistic testing were performed on the selected phytochemicals and the positive control antibiotic, amoxicillin. The in-silico study revealed that oroxindin, rosmarinic acid and verbascoside are inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase, respectively, in which, oroxindin has the highest potency against H. pylori, indicated by a minimum inhibitory concentration (MIC) value of 50 µg/mL. A combination of oroxindin and amoxicillin demonstrated additive effects against H. pylori, as indicated by a fractional inhibitory concentration (FIC) value of 0.75. This study identified phytochemicals that deserve further investigation for the development of adjuvant therapeutic agents to current antibiotics against H. pylori.

[Label-free small molecule probe and target discovery of traditional Chinese medicine].

Target discovery is the core of elucidating the mechanism of traditional Chinese medicine( TCM),and it is also the key to correlate the chemical composition and pharmacological action of TCM. The traditional target screening methods such as the activitybased probe profiling,affinity chromatography,and protein microarray are commonly used in the past,however,which are limited in TCM due to the complexity of small molecules existed in the herbal medicine. The label-free small molecule probe is a recently well-applied technology in the target discovery of natural products,which is characterized by discovering the small molecule-protein ligation without any structural modification at the ligands,and is therefore suitable to the complex chemical constituents in TCM. Furthermore,this method is conducted on the basis of proteome,which is advanced in the discovery of new or multiple target proteins of TCM. Owing to the potential of label-free probe in the target discovery of TCM,its analytical principle,application status,and general protocol were reviewed in this paper. The label-free probe technology is anticipated to accelerate the mechanism-uncovering of TCM.

Physiological relevance of covalent protein modification by dietary isothiocyanates.

Isothiocyanates (ITCs), naturally occurring in abundance in cruciferous vegetables, are the most well-studied organosulfur compounds having an electrophilic reactivity. ITCs have been accepted as major ingredients of these vegetables that afford their health promoting potentials. ITCs are able to modulate protein functions related to drug-metabolizing enzymes, transporters, kinases and phosphatases, etc. One of the most important questions about the molecular basis for the health promoting effects of ITCs is how they modulate cellular target proteins. Although the molecular targets of ITCs remains to be validated, dietary modulation of the target proteins via covalent modification by ITCs should be one of the promising strategies for the protection of cells against oxidative and inflammatory damage. This review discusses the plausible target proteins of dietary ITCs with an emphasis on possible involvement of protein modification in their health promoting effects. The fundamental knowledge of ITCs is also included with consideration of the chemistry, intracellular behavior, and metabolism.

Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

BACKGROUND: Allergic reactions to ß-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. METHODS: We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. RESULTS: We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. CONCLUSIONS: These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response.

The proteome targets of intracellular targeting antimicrobial peptides.

Antimicrobial peptides have been considered well-deserving candidates to fight the battle against microorganisms due to their broad-spectrum antimicrobial activities. Several studies have suggested that membrane disruption is the basic mechanism of AMPs that leads to killing or inhibiting microorganisms. Also, AMPs have been reported to interact with macromolecules inside the microbial cells such as nucleic acids (DNA/RNA), protein synthesis, essential enzymes, membrane septum formation and cell wall synthesis. Proteins are associated with many intracellular mechanisms of cells, thus protein targets may be specifically involved in mechanisms of action of AMPs. AMPs like pyrrhocoricin, drosocin, apidecin and Bac 7 are documented to have protein targets, DnaK and GroEL. Moreover, the intracellular targeting AMPs are reported to influence more than one protein targets inside the cell, suggesting for the multiple modes of actions. This complex mechanism of intracellular targeting AMPs makes them more difficult for the development of resistance. Herein, we have summarized the current status of AMPs in terms of their mode of actions, entry to cytoplasm and inhibition of macromolecules. To reveal the mechanism of action, we have focused on AMPs with intracellular protein targets. We have also included the use of high-throughput proteome microarray to determine the unidentified AMP protein targets in this review.

Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification.

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.

Expression of the moss PpLEA4-20 gene in rice enhances membrane protection and client proteins stability.

Green vegetative tissues of the moss Physcomitrella patens possess a powerful ability to tolerate severe drought stress. Proteomics analysis have revealed that a large number of late embryogenesis abundant (LEA) proteins were key players in the drought tolerance of the photosynthetic tissues. PpLEA4-20, a member of the moss LEA protein family, was selected for further function study using an ectopic expression method in rice. Through molecular identification via PCR, southern blotting and TAIL-PCR, we demonstrated that the PpLEA4-20 gene was transformed and inserted into a non-encoded region in chromosome 4 of rice and expressed stably in transgenic rice. Unexpectedly, PpLEA4-20 protein emerged as two high-expressed spots on 2-D gels generated from transgenic rice, suggesting that PpLEA4-20 proteins are complete compatible and might be modified in rice. Both growth and physiological analysis showed that seedlings of transgenic PpLEA4-20 rice displayed altered phenotypes and tolerance to salt. In addition, electrolyte leakage was reduced in transgenic PpLEA4-20 compared to wild type under stress conditions. Anti-aggregation analysis found that the PpLEA4-20 protein expressed in rice remained soluble at high temperature and in addition to some native proteins from transgenic PpLEA4-20 rice. Based on Nano LC MS/MS analysis, we identified several proteins from transgenic PpLEA4-20 rice of increased heat-stability. Our results provide evidence for a role of PpLEA4-20 in salt tolerance and stabilization of client proteins.

CANDI: A Web Server for Predicting Molecular Targets and Pathways of Cannabis-Based Therapeutics.

Background: Cannabis sativa with a rich history of traditional medicinal use, has garnered significant attention in contemporary research for its potential therapeutic applications in various human diseases, including pain, inflammation, cancer, and osteoarthritis. However, the specific molecular targets and mechanisms underlying the synergistic effects of its diverse phytochemical constituents remain elusive. Understanding these mechanisms is crucial for developing targeted, effective cannabis-based therapies. Methods: To investigate the molecular targets and pathways involved in the synergistic effects of cannabis compounds, we utilized DRIFT, a deep learning model that leverages attention-based neural networks to predict compound-target interactions. We considered both whole plant extracts and specific plant-based formulations. Predicted targets were then mapped to the Reactome pathway database to identify the biological processes affected. To facilitate the prediction of molecular targets and associated pathways for any user-specified cannabis formulation, we developed CANDI (Cannabis-derived compound Analysis and Network Discovery Interface), a web-based server. This platform offers a user-friendly interface for researchers and drug developers to explore the therapeutic potential of cannabis compounds. Results: Our analysis using DRIFT and CANDI successfully identified numerous molecular targets of cannabis compounds, many of which are involved in pathways relevant to pain, inflammation, cancer, and other diseases. The CANDI server enables researchers to predict the molecular targets and affected pathways for any specific cannabis formulation, providing valuable insights for developing targeted therapies. Conclusions: By combining computational approaches with knowledge of traditional cannabis use, we have developed the CANDI server, a tool that allows us to harness the therapeutic potential of cannabis compounds for the effective treatment of various disorders. By bridging traditional pharmaceutical development with cannabis-based medicine, we propose a novel approach for botanical-based treatment modalities.

Aptamer-based sensors for fluid biopsies of protein disease markers.

Fluid biopsy technology, characterized by its minimally invasive nature, speed, and continuity, has become a rapidly advancing and widely applied real-time diagnostic technique. Among various biomarkers, proteins represent the most abundant class of disease indicators. The sensitive and accurate detection of protein markers in bodily fluids is significantly influenced by the control exerted by recognition ligands. Aptamers, which are structurally dynamic functional oligonucleotides, exhibit high affinity, specific recognition of targets, and notable characteristics of high editability and modularity. These features make aptamer universal "recognition-capture" components, contribute to a significant leap in their applications within the biosensor domain. In this context, we provide a comprehensive review of the extensive application of aptamer-based biosensors in fluid biopsy. We systematically compile the characteristics and construction strategies of aptamer-based biosensors tailored for fluid biopsy, including aptamer sequences, affinity (KD), fluid background, sensing technologies, sensor construction strategies, incubation time, detection performance, and influencing factors. Furthermore, a comparative analysis of their advantages and disadvantages was conducted. In conclusion, we delineate and deliberate on prospective research trajectories and challenges that lie ahead in the realm of aptamer-based biosensors for fluid biopsy.

Comparative Metabolomics and Network Pharmacology Analysis Reveal Shared Neuroprotective Mechanisms of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb.

Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., two nootropics, are recognized in Indian Ayurvedic texts. Studies have attempted to understand their action as memory enhancers and neuroprotectants, but many molecular aspects remain unknown. We propose that Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb. share common neuroprotective mechanisms. Mass spectrometry-based untargeted metabolomics and network pharmacology approach were used to identify potential protein targets for the metabolites from each extract. Phytochemical analyses and cell culture validation studies were also used to assess apoptosis and ROS activity using aqueous extracts prepared from both herbal powders. Further, docking studies were also performed using the LibDock protocol. Untargeted metabolomics and network pharmacology approach unveiled 2751 shared metabolites and 3439 and 2928 non-redundant metabolites from Bacopa monnieri and Centella asiatica extracts, respectively, suggesting a potential common neuroprotective mechanism among these extracts. Protein-target prediction highlighted 92.4% similarity among the proteins interacting with metabolites for these extracts. Among them, kinases mapped to MAPK, mTOR, and PI3K-AKT signaling pathways represented a predominant population. Our results highlight a significant similarity in the metabolome of Bacopa monnieri (L.) Wettst and Centella asiatica (L.) Urb., and their potential protein targets may be attributed to their common neuroprotective functions.

Prediction of Protein Targets in Ovarian Cancer Using a Ru-Complex and Carbon Dot Drug Delivery Therapeutic Nanosystems: A Bioinformatics and µ-FTIR Spectroscopy Approach.

We predicted the protein therapeutic targets specific to a Ru-based potential drug and its combination with pristine and N-doped carbon dot drug delivery systems, denoted as RuCN/CDs and RuCN/N-CDs. Synchrotron-based FTIR microspectroscopy (µFTIR) in addition to bioinformatics data on drug structures and protein sequences were applied to assess changes in the protein secondary structure of A2780 cancer cells. µFTIR revealed the moieties of the target proteins' secondary structure changes only after the treatment with RuCN and RuCN/N-CDs. A higher content of α-helices and a lower content of ß-sheets appeared in A2780 cells after RuCN treatment. Treatment with RuCN/N-CDs caused a substantial increase in parallel ß-sheet numbers, random coil content, and tyrosine residue numbers. The results obtained suggest that the mitochondrion-related proteins NDUFA1 and NDUFB5 are affected by RuCN either via overexpression or stabilisation of helical structures. RuCN/N-CDs either induce overexpression of the ß-sheet-rich protein NDUFS1 and affect its random coil structure or interact and stabilise its structure via hydrogen bonding between -NH2 groups from N-CDs with protein C=O groups and -OH groups of serine, threonine, and tyrosine residues. The N-CD nanocarrier tunes this drug's action by directing it toward a specific protein target, changing this drug's coordination ability and inducing changes in the protein's secondary structures and function.

Recent advances in identifying protein targets of bioactive natural products.

Background: Natural products exhibit structural complexity, diversity, and historical therapeutic significance, boasting attractive functions and biological activities that have significantly influenced drug discovery endeavors. The identification of target proteins of active natural compounds is crucial for advancing novel drug innovation. Currently, methods for identifying targets of natural products can be categorized into labeling and label-free approaches based on whether the natural bioactive constituents are modified into active probes. In addition, there is a new avenue for rapidly exploring the targets of natural products based on their innate functions. Aim: This review aimed to summarize recent advancements in both labeling and label-free approaches to the identification of targets for natural products, as well as the novel target identification method based on the natural functions of natural products. Methods: We systematically collected relevant articles published in recent years from PubMed, Web of Science, and ScienceDirect, focusing on methods employed for identifying protein targets of bioactive natural products. Furthermore, we systematically summarized the principles, procedures, and successful cases, as well as the advantages and limitations of each method. Results: Labeling methods allow for the direct labeling of target proteins and the exclusion of indirectly targeted proteins. However, these methods are not suitable for studying post-modified compounds with abolished activity, chemically challenging synthesis, or trace amounts of natural active compounds. Label-free methods can be employed to identify targets of any natural active compounds, including trace amounts and multicomponent mixtures, but their reliability is not as high as labeling methods. The structural complementarity between natural products and their innate receptors significantly increase the opportunities for finding more promising structural analogues of the natural products, and natural products may interact with several structural analogues of receptors in humans. Conclusion: Each approach presents benefits and drawbacks. In practice, a combination of methods is employed to identify targets of natural products. And natural products' innate functions-based approach is a rapid and selective strategy for target identification. This review provides valuable references for future research in this field, offering insights into techniques and methodologies.