Application of PROTACs in Target Identification and Target Validation.

PROTAC, as a novel therapeutic drug model, has received widespread attention from the academic and pharmaceutical industries. At the same time, PROTAC technology has led many researchers to focus on developing chemical biology tool properties due to its unique operating mechanism and protein dynamic regulatory properties. In recent years, the rapid development of PROTAC technology has gradually made it an essential tool for target identification and target validation. To further promote the application of PROTAC tools in drug discovery and basic medical sciences research, this review distinguished between target identification and target validation concepts. It summarized the research progress of PROTAC technology in these aspects.

Discovery of GuaB inhibitors with efficacy against Acinetobacter baumannii infection.

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.

Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery.

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.

CircRNAs in Pancreatic Cancer: New Tools for Target Identification and Therapeutic Intervention.

We have reviewed the literature for circular RNAs (circRNAs) with efficacy in preclinical pancreatic-cancer related in vivo models. The identified circRNAs target chemoresistance mechanisms (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), components of the signaling- (n=11), ubiquitination- (n=2), autophagy-system (n=2), and others (n=9). In addition to identifying targets for therapeutic intervention, circRNAs are potential new entities for treatment of pancreatic cancer. Up-regulated circRNAs can be inhibited by antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or clustered regularly interspaced short-palindromic repeats-CRISPR associated protein (CRISPR-CAS)-based intervention. The function of down-regulated circRNAs can be reconstituted by replacement therapy using plasmids or virus-based vector systems. Target validation experiments and the development of improved delivery systems for corresponding agents were examined.

Identification of potent indolizine derivatives against Mycobacterial tuberculosis: In vitro anti-TB properties, in silico target validation, molecular docking and dynamics studies.

In the current study, two sets of compounds: (E)-1-(2-(4-substitutedphenyl)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium derivatives (3a-3e); and (E)-3-(substitutedbenzoyl)-7-((hydroxyimino)methyl)-2-substitutedindolizine-1-carboxylate derivatives (5a-5j), were synthesized and biologically evaluated against two strains of Mycobacterial tuberculosis (ATCC 25177) and multi-drug resistant (MDR) strains. Further, they were also tested in vitro against the mycobacterial InhA enzyme. The in vitro results showed excellent inhibitory activities against both MTB strains and compounds 5a-5j were found to be more potent, and their MIC values ranged from 5 to 16 µg/mL and 16-64 µg/mL against the M. tuberculosis (ATCC 25177) and MDR-TB strains, respectively. Compound 5h with phenyl and 4-fluorobenzoyl groups attached to the 2- and 3-position of the indolizine core was found to be the most active against both strains with MIC values of 5 µg/mL and 16 µg/mL, respectively. On the other hand, the two sets of compounds showed weak to moderate inhibition of InhA enzyme activity that ranged from 5 to 17 % and 10-52 %, respectively, with compound 5f containing 4-fluoro benzoyl group attached to the 3-position of the indolizine core being the most active (52 % inhibition of InhA). Unfortunately, there was no clear correlation between the InhA inhibitory activity and MIC values of the tested compounds, indicating the probability that they might have different modes of action other than InhA inhibition. Therefore, a computational investigation was conducted by employing molecular docking to identify their putative drug target(s) and, consequently, understand their mechanism of action. A panel of 20 essential mycobacterial enzymes was investigated, of which ß-ketoacyl acyl carrier protein synthase I (KasA) and pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (BioA) enzymes were revealed as putative targets for compounds 3a-3e and 5a-5j, respectively. Moreover, in silico ADMET predictions showed adequate properties for these compounds, making them promising leads worthy of further optimization.

A key regulator of male gametogenesis in Cryptosporidium.

Gamete development is a precisely programmed process in Cryptosporidium parvum, a leading cause of diarrheal disease worldwide. Nava et al. recently described the developmentally regulated expression of CDPK5 during male gametogenesis. Here we discuss their main findings, posing this protein kinase as a promising target for antiparasitic interventions.

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

ST-2560, a selective inhibitor of the NaV1.7 sodium channel, affects nocifensive and cardiovascular reflexes in non-human primates.

BACKGROUND AND PURPOSE: The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. EXPERIMENTAL APPROACH: Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. KEY RESULTS: ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. CONCLUSIONS AND IMPLICATIONS: Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.

Sarmentol H derived from Sedum sarmentosum Bunge directly targets FXR to mitigate cholestasis by recruiting SRC-1.

BACKGROUND: Farnesoid X receptor (FXR) is a vital receptor for bile acids and plays an important role in the treatment of cholestatic liver disease. In addition to traditional bile acid-based steroidal agonists, synthetic alkaloids are the most commonly reported non-steroidal FXR agonists. Sarmentol H is a nor-sesquiterpenoid obtained from Sedum sarmentosum Bunge, and in vitro screening experiments have shown that it might be related to the regulation of the FXR pathway in a previous study. PURPOSE: To investigate the therapeutic effects of sarmentol H on cholestasis and to determine whether sarmentol H directly targets FXR to mitigate cholestasis. Furthermore, this study aimed to explore the key amino acid residues involved in the binding of sarmentol H to FXR through site-directed mutagenesis. METHODS: An intrahepatic cholestasis mouse model was established to investigate the therapeutic effects of sarmentol H on cholestasis. In vitro experiments, including Co-Ip and FXR-EcRE-Luc assays, were performed to assess whether sarmentol H activates FXR by recruiting the receptor coactivator SRC1. CETSA, SIP, DARTS, and ITC were used to determine the binding of sarmentol H to FXR protein. The key amino acid residues for sarmentol H binding to FXR were analyzed by molecular docking and site-directed mutagenesis. Finally, we conducted in vivo experiments on wild-type and Fxr-/- mice to further validate the anticholestatic target of sarmentol H. RESULTS: Sarmentol H had significant ameliorative effects on the pathological conditions of cholestatic mice induced with ANIT. In vitro experiments suggested that it is capable of activating FXR and regulating downstream signaling pathways by recruiting SRC1. The target validation experiments showed that sarmentol H had the ability to bind to FXR as a ligand (KD = 2.55 µmol/L) and enhance the stability of its spatial structure. Moreover, site-directed mutagenesis revealed that THR292 and TYR365 were key binding sites for sarmentol H and FXR. Furthermore, knockout of the Fxr gene resulted in a significantly higher degree of ANIT-induced cholestatic liver injury than that in wild-type cholestatic mice, and the amelioration of cholestasis or regulatory effects on FXR downstream genes by sarmentol H also disappeared in Fxr-/- cholestatic mice. CONCLUSION: Sarmentol H is an FXR agonist. This is the first study to show that it exerts a significant therapeutic effect on cholestatic mice, and can directly bind to FXR and activate it by recruiting the coactivator SRC1.

ZebraReg-a novel platform for discovering regulators of cardiac regeneration using zebrafish.

Cardiovascular disease is the leading cause of death worldwide with myocardial infarction being the most prevalent. Currently, no cure is available to either prevent or revert the massive death of cardiomyocytes that occurs after a myocardial infarction. Adult mammalian hearts display a limited regeneration capacity, but it is insufficient to allow complete myocardial recovery. In contrast, the injured zebrafish heart muscle regenerates efficiently through robust proliferation of pre-existing myocardial cells. Thus, zebrafish allows its exploitation for studying the genetic programs behind cardiac regeneration, which may be present, albeit dormant, in the adult human heart. To this end, we have established ZebraReg, a novel and versatile automated platform for studying heart regeneration kinetics after the specific ablation of cardiomyocytes in zebrafish larvae. In combination with automated heart imaging, the platform can be integrated with genetic or pharmacological approaches and used for medium-throughput screening of presumed modulators of heart regeneration. We demonstrate the versatility of the platform by identifying both anti- and pro-regenerative effects of genes and drugs. In conclusion, we present a tool which may be utilised to streamline the process of target validation of novel gene regulators of regeneration, and the discovery of new drug therapies to regenerate the heart after myocardial infarction.

Degron tagging for rapid protein degradation in mice.

Degron tagging allows proteins of interest to be rapidly degraded, in a reversible and tuneable manner, in response to a chemical stimulus. This provides numerous opportunities for understanding disease mechanisms, modelling therapeutic interventions and constructing synthetic gene networks. In recent years, many laboratories have applied degron tagging successfully in cultured mammalian cells, spurred by rapid advances in the fields of genome editing and targeted protein degradation. In this At a Glance article, we focus on recent efforts to apply degron tagging in mouse models, discussing the distinct set of challenges and opportunities posed by the in vivo environment.

Deregulated circRNAs in Epithelial Ovarian Cancer With Activity in Preclinical In Vivo Models: Identification of Targets and New Modalities for Therapeutic Intervention.

Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

Prioritising genetic findings for drug target identification and validation.

The decreasing costs of high-throughput genetic sequencing and increasing abundance of sequenced genome data have paved the way for the use of genetic data in identifying and validating potential drug targets. However, the number of identified potential drug targets is often prohibitively large to experimentally evaluate in wet lab experiments, highlighting the need for systematic approaches for target prioritisation. In this review, we discuss principles of genetically guided drug development, specifically addressing loss-of-function analysis, colocalization and Mendelian randomisation (MR), and the contexts in which each may be most suitable. We subsequently present a range of biomedical resources which can be used to annotate and prioritise disease-associated proteins identified by these studies including 1) ontologies to map genes, proteins, and disease, 2) resources for determining the druggability of a potential target, 3) tissue and cell expression of the gene encoding the potential target, and 4) key biological pathways involving the potential target. We illustrate these concepts through a worked example, identifying a prioritised set of plasma proteins associated with non-alcoholic fatty liver disease (NAFLD). We identified five proteins with strong genetic support for involvement with NAFLD: CYB5A, NT5C, NCAN, TGFBI and DAPK2. All of the identified proteins were expressed in both liver and adipose tissues, with TGFBI and DAPK2 being potentially druggable. In conclusion, the current review provides an overview of genetic evidence for drug target identification, and how biomedical databases can be used to provide actionable prioritisation, fully informing downstream experimental validation.

Discovery of a Small Molecule Activator of Slack (Kcnt1) Potassium Channels That Significantly Reduces Scratching in Mouse Models of Histamine-Independent and Chronic Itch.

Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

Platanosides from Platanus × acerifolia: New molecules, SAR, and target validation of a strong lead for drug-resistant bacterial infections and the associated sepsis.

Three undescribed (1-3) and nine known (4-12) platanosides were isolated and characterized from a bioactive extract of the May leaves of Platanus × acerifolia that initially showed inhibition against Staphylococcus aureus. Targeted compound mining was guided by an LC-MS/MS-based molecular ion networking (MoIN) strategy combined with conventional isolation procedures from a unique geographic location. The novel structures were mainly determined by 2D NMR and computational (NMR/ECD calculations) methods. Compound 1 is a rare acylated kaempferol rhamnoside possessing a truxinate unit. 6 (Z,E-platanoside) and 7 (E,E-platanoside) were confirmed to have remarkable inhibitory effects against both methicillin-resistant S. aureus (MIC: ≤ 16 µg/mL) and glycopeptide-resistant Enterococcus faecium (MIC: ≤ 1 µg/mL). These platanosides were subjected to docking analyses against FabI (enoyl-ACP reductase) and PBP1/2 (penicillin binding protein), both of which are pivotal enzymes governing bacterial growth but not found in the human host. The results showed that 6 and 7 displayed superior binding affinities towards FabI and PBP2. Moreover, surface plasmon resonance studies on the interaction of 1/7 and FabI revealed that 7 has a higher affinity (KD = 1.72 µM), which further supports the above in vitro data and is thus expected to be a novel anti-antibacterial drug lead.

Novel Proline Transporter Inhibitor (LQFM215) Presents Antipsychotic Effect in Ketamine Model of Schizophrenia.

The glutamatergic hypothesis of schizophrenia suggests a correlation between NMDA receptor hypofunction and negative psychotic symptoms. It has been observed that the expression of the proline transporter (PROT) in the central nervous system (CNS) is associated with glutamatergic neurotransmission, as L-proline has the capacity to activate and modulate AMPA and NMDA receptors. In this study, we aimed to investigate whether inhibition of proline transporters could enhance glutamatergic neurotransmission and potentially exhibit antipsychotic effects in an experimental schizophrenia model. Using molecular dynamics analysis in silico, we validated an innovative PROT inhibitor, LQFM215. We quantified the cytotoxicity of LQFM215 in the Lund human mesencephalic cell line (LUHMES). Subsequently, we employed the ketamine-induced psychosis model to evaluate the antipsychotic potential of the inhibitor, employing behavioral tests including open-field, three-chamber interaction, and prepulse inhibition (PPI). Our results demonstrate that LQFM215, at pharmacologically active concentrations, exhibited negligible neurotoxicity when astrocytes were co-cultured with neurons. In the ketamine-induced psychosis model, LQFM215 effectively reduced hyperlocomotion and enhanced social interaction in a three-chamber social approach task across all administered doses. Moreover, the compound successfully prevented the ketamine-induced disruption of sensorimotor gating in the PPI test at all tested doses. Overall, these findings suggest that PROT inhibition could serve as a potential therapeutic target for managing symptoms of schizophrenia model.

The discovery of a new potent FXR agonist based on natural product screening.

FXR agonistic activity screening was conducted based on natural product resources containing 38 structurally diverse sesquiterpenoids isolated from Xylopia vielana. Among them, 34 undescribed sesquiterpenoids with 5 different skeleton types were first characterized by HRESIMS, NMR data, ECD calculations and X-ray crystallographic analysis. High-content screening for FXR agonistic activity of these compounds demonstrated that 13 compounds could activate FXR. Then molecular docking results suggested that hydrogen bonding and hydrophobic interactions might contribute to the main interaction of active compounds with FXR. The preliminary structure-activity relationships (SARs) of those isolates were also discussed. The most potent compound 27 significantly elevated the transcriptional activity of the FXR target gene BSEP promoter (EC50 = 14.26 µM) by a dual-luciferase reporter assay. Western blotting indicated that compound 27 activated the FXR-associated pathway, thereby upregulating SHP and BSEP expression, and downregulating CYP7A1 and NTCP expression. We further revealed that FXR was the target protein of compound 27 through diverse target validation methods, including CETSA, SIP, and DARTS under the intervention of temperature, organic reagents and protease. Pharmacological in vivo experiments showed that compound 27 effectively ameliorated α-naphthyl isothiocyanate (ANIT)-induced cholestasis in mice, as evidenced by the ameliorative histopathology of the liver and the decrease in biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA). This work showed a practical strategy for the discovery of new FXR agonists from natural products and provided potential insights for sesquiterpenoids as FXR agonist lead compounds.

Chemical Probes Review: Choosing the Right Path Towards Pharmacological Targets in Drug Discovery, Challenges and Future Perspectives.

Chemical probes are essential for academic research and target validation for disease identification. They facilitate drug discovery, target function investigation, and translation studies. A chemical probe provides starting material that can accelerate therapeutic values and safety measures for identifying any biological target in drug discovery. Essential read outs depend on their versatility in biochemical testing, proving the hypothesis, selectivity, specificity, affinity towards the target site, and valuable in new therapeutic approaches. Disease management will depend upon chemical probes as a primitive tool to ascertain the physicochemical stability for in vivo and in vitro studies useful for clinical trials and industrial application in the future. For cancer research, bacterial infection, and neurodegenerative disorders, chemical probes are integrated circuits which are on pipeline for the drug discovery process Furthermore, pharmacological modulators incorporate activators, crosslinkers, degraders, and inhibitors. Reports accessed depend on their structural, mechanical, biochemical, and pharmacological characterization in drug discovery research. The perspective for designing any chemical probes concludes with the utilization of drug discovery and identification of the potential target. It focuses mainly on evidence-based studies and produces promising results in successfully delivering novel therapeutics to treat cancers and other disorders at the target site. Moreover, natural product pharmacophores like rapamycin, cephalosporin, and ß-lactamase are utilized for drug discovery. Chemical probes revolutionize computational-based study design depending on identifying novel targets within the database framework. Chemical probes are the clinical answers for drug development and goforward tools in solving other riddles for scientists and researchers working in this industries.