Biophysical Assays for Investigating Modulators of Macromolecular Complexes: An Overview.

Drug discovery is a lengthy and intricate process, and in its early stage, crucial steps are the selection of the therapeutic target and the identification of novel ligands. Most targets are dysregulated in pathogenic cells; typically, their activation or deactivation leads to the desired effect, while in other cases, interfering with the target-natural binder complex achieves the therapeutic results. Biophysical assays are a suitable strategy for finding new ligands or interferent agents, being able to evaluate ligand-protein interactions and assessing the effect of small molecules (SMols) on macromolecular complexes. This mini-review provides a detailed analysis of widely used biophysical methods, including fluorescence-based approaches, circular dichroism, isothermal titration calorimetry, microscale thermophoresis, and NMR spectroscopy. After a brief description of the methodologies, examples of interaction and competition experiments are described, together with an analysis of the advantages and disadvantages of each technique. This mini-review provides an overview of the most relevant biophysical technologies that can help in identifying SMols able not only to bind proteins but also to interfere with macromolecular complexes.

Neurodegeneration: can metabolites from Eremurus persicus help?

The number of patients affected by neurodegenerative diseases is increasing worldwide, and no effective treatments have been developed yet. Although precision medicine could represent a powerful tool, it remains a challenge due to the high variability among patients. To identify molecules acting with innovative mechanisms of action, we performed a computational investigation using SAFAN technology, focusing specifically on HuD. This target belongs to the human embryonic lethal abnormal visual-like (ELAV) proteins and plays a key role in neuronal plasticity and differentiation. The results highlighted that the molecule able to bind the selected target was (R)-aloesaponol-III-8-methyl ether [(R)-ASME], a metabolite extracted from Eremurus persicus. Notably, this molecule is a TNF-α inhibitor, a cytokine involved in neuroinflammation. To obtain a suitable amount of (R)-ASME to confirm its activity on HuD, we optimized the extraction procedure. Together with ASME, another related metabolite, germichrysone, was isolated. Both ASME and germichrysone underwent biological investigation, but only ASME confirmed its ability to bind HuD. Given the multifactorial nature of neurodegenerative diseases, we decided to investigate ASME as a proteasome activator, being molecules endowed with this kind of activity potentially able to counteract aggregations of dysregulated proteins. ASME was able to activate the considered target both in enzymatic and cellular assays. Therefore, ASME may be considered a promising hit in the fight against neurodegenerative diseases.

The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents.

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

Sigma receptor and aquaporin modulators: chiral resolution, configurational assignment, and preliminary biological profile of RC752 enantiomers.

The key role of chiral small molecules in drug discovery programs has been deeply investigated throughout last decades. In this context, our previous studies highlighted the influence of the absolute configuration of different stereocenters on the pharmacokinetic, pharmacodynamic and functional properties of promising Sigma receptor (SR) modulators. Thus, starting from the racemic SR ligand RC752, we report herein the isolation of the enantiomers via enantioselective separation with both HPLC and SFC. After optimization of the eco-sustainable chiral SFC method, both enantiomers were obtained in sufficient amount (tens of mg) and purity (ee up to 95%) to allow their characterization and initial biological investigation. Both enantiomers a) displayed a high affinity for the S1R subtype (Ki = 15.0 ± 1.7 and 6.0 ± 1.2 nM for the (S)- and (R)-enantiomer, respectively), but only negligible affinity toward the S2R (> 350 nM), and b) were rapidly metabolized when incubated with mouse and human hepatic microsomes. Furthermore, the activity on AQP-mediated water permeability indicated a different functional profile for the enantiomers in terms of modulatory effect on the peroxiporins gating.

Identification of HuR-RNA Interfering Compounds by Dynamic Combinatorial Chemistry and Fluorescence Polarization.

The RNA binding protein HuR regulates the post-transcriptional process of different oncogenes and tumor suppressor genes, and its dysregulation is linked with cancer. Thus, modulating the complex HuR-RNA represents a promising anticancer strategy. To search for novel HuR ligands able to interfere with the HuR-RNA complex, the protein-templated dynamic combinatorial chemistry (pt-DCC) method was utilized. The recombinant RRM1+2 protein construct, which contains essential domains for ligand-HuR binding and exhibits enhanced solubility and stability compared to the native protein, was used for pt-DCC. Seven acylhydrazones with over 80% amplification were identified. The binding of the fragments to HuR extracted from DCC was validated using STD-NMR, and molecular modeling studies revealed the ability of the compounds to bind HuR at the mRNA binding pocket. Notably, three compounds effectively interfered with HuR-RNA binding in fluorescence polarization studies, suggesting their potential as foundational compounds for developing anticancer HuR-RNA interfering agents.

High-Throughput Phenotypic Screening and Machine Learning Methods Enabled the Selection of Broad-Spectrum Low-Toxicity Antitrypanosomatidic Agents.

Broad-spectrum anti-infective chemotherapy agents with activity against Trypanosomes, Leishmania, and Mycobacterium tuberculosis species were identified from a high-throughput phenotypic screening program of the 456 compounds belonging to the Ty-Box, an in-house industry database. Compound characterization using machine learning approaches enabled the identification and synthesis of 44 compounds with broad-spectrum antiparasitic activity and minimal toxicity against Trypanosoma brucei, Leishmania Infantum, and Trypanosoma cruzi. In vitro studies confirmed the predictive models identified in compound 40 which emerged as a new lead, featured by an innovative N-(5-pyrimidinyl)benzenesulfonamide scaffold and promising low micromolar activity against two parasites and low toxicity. Given the volume and complexity of data generated by the diverse high-throughput screening assays performed on the compounds of the Ty-Box library, the chemoinformatic and machine learning tools enabled the selection of compounds eligible for further evaluation of their biological and toxicological activities and aided in the decision-making process toward the design and optimization of the identified lead.

Peptides as Therapeutic Agents: Challenges and Opportunities in the Green Transition Era.

Peptides are at the cutting edge of contemporary research for new potent, selective, and safe therapeutical agents. Their rise has reshaped the pharmaceutical landscape, providing solutions to challenges that traditional small molecules often cannot address. A wide variety of natural and modified peptides have been obtained and studied, and many others are advancing in clinical trials, covering multiple therapeutic areas. As the demand for peptide-based therapies grows, so does the need for sustainable and environmentally friendly synthesis methods. Traditional peptide synthesis, while effective, often involves environmentally draining processes, generating significant waste and consuming vast resources. The integration of green chemistry offers sustainable alternatives, prioritizing eco-friendly processes, waste reduction, and energy conservation. This review delves into the transformative potential of applying green chemistry principles to peptide synthesis by discussing relevant examples of the application of such approaches to the production of active pharmaceutical ingredients (APIs) with a peptide structure and how these efforts are critical for an effective green transition era in the pharmaceutical field.

Could the Oxidation of α1-Antitrypsin Prevent the Binding of Human Neutrophil Elastase in COVID-19 Patients?

Human neutrophil elastase (HNE) is involved in SARS-CoV-2 virulence and plays a pivotal role in lung infection of patients infected by COVID-19. In healthy individuals, HNE activity is balanced by α1-antitrypsin (AAT). This is a 52 kDa glycoprotein, mainly produced and secreted by hepatocytes, that specifically inhibits HNE by blocking its activity through the formation of a stable complex (HNE-AAT) in which the two proteins are covalently bound. The lack of this complex, together with the detection of HNE activity in BALf/plasma samples of COVID-19 patients, leads us to hypothesize that potential functional deficiencies should necessarily be attributed to possible structural modifications of AAT. These could greatly diminish its ability to inhibit neutrophil elastase, thus reducing lung protection. The aim of this work was to explore the oxidation state of AAT in BALf/plasma samples from these patients so as to understand whether the deficient inhibitory activity of AAT was somehow related to possible conformational changes caused by the presence of abnormally oxidized residues.

Discovery of potent pyrrolo-pyrimidine and purine HDAC inhibitors for the treatment of advanced prostate cancer.

The development of drugs for the treatment of advanced prostate cancer (PCA) remains a challenging task. In this study we have designed, synthesized and tested twenty-nine novel HDAC inhibitors based on three different zinc binding groups (trifluoromethyloxadiazole, hydroxamic acid, and 2-mercaptoacetamide). These warheads were conveniently tethered to variously substituted phenyl linkers and decorated with differently substituted pyrrolo-pyrimidine and purine cap groups. Remarkably, most of the compounds showed nanomolar inhibitory activity against HDAC6. To provide structural insights into the Structure-Activity Relationships (SAR) of the investigated compounds, docking of representative inhibitors and molecular dynamics of HDAC6-inhibitor complexes were performed. Compounds of the trifluoromethyloxadiazole and hydroxamic acid series exhibited promising anti-proliferative activities, HDAC6 targeting in PCA cells, and in vitro tumor selectivity. Representative compounds of the two series were tested for solubility, cell permeability and metabolic stability, demonstrating favorable in vitro drug-like properties. The more interesting compounds were subjected to migration assays, which revealed that compound 13 and, to a lesser extent, compound 15 inhibited the invasive behaviour of androgen-sensitive and -insensitive advanced prostate cancer cells. Compound 13 was profiled against all HDACs and found to inhibit all members of class II HDACs (except for HDAC10) and to be selective with respect to class I and class IV HDACs. Overall, compound 13 combines potent inhibitory activity and class II selectivity with favorable drug-like properties, an excellent anti-proliferative activity and marked anti-migration properties on PCA cells, making it an excellent lead candidate for further optimization.

Short- and Long-Term Regulation of HuD: A Molecular Switch Mediated by Folic Acid?

The RNA-binding protein HuD has been shown to play a crucial role in gene regulation in the nervous system and is involved in various neurological and psychiatric diseases. In this study, through the creation of an interaction network on HuD and its potential targets, we identified a strong association between HuD and several diseases of the nervous system. Specifically, we focused on the relationship between HuD and the brain-derived neurotrophic factor (BDNF), whose protein is implicated in several neuronal diseases and is involved in the regulation of neuronal development, survival, and function. To better investigate this relationship and given that we previously demonstrated that folic acid (FA) is able to directly bind HuD itself, we performed in vitro experiments in neuron-like human SH-SY5Y cells in the presence of FA, also known to be a pivotal environmental factor influencing the nervous system development. Our findings show that FA exposure results in a significant increase in both HuD and BDNF transcripts and proteins after 2 and 4 h of treatment, respectively. Similar data were obtained after 2 h of FA incubation followed by 2 h of washout. This increase was no longer detected upon 24 h of FA exposure, probably due to a signaling shutdown mechanism. Indeed, we observed that following 24 h of FA exposure HuD is methylated. These findings indicate that FA regulates BDNF expression via HuD and suggest that FA can behave as an epigenetic modulator of HuD in the nervous system acting via short- and long-term mechanisms. Finally, the present results also highlight the potential of BDNF as a therapeutic target for specific neurological and psychiatric diseases.

Discovery of RC-752, a Novel Sigma-1 Receptor Antagonist with Antinociceptive Activity: A Promising Tool for Fighting Neuropathic Pain.

Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world's population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Identification of a novel extracellular inhibitor of FGF2/FGFR signaling axis by combined virtual screening and NMR spectroscopy approach.

The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity.

Structural Insights into the Ligand-LsrK Kinase Binding Mode: A Step Forward in the Discovery of Novel Antimicrobial Agents.

LsrK is a bacterial kinase that triggers the quorum sensing, and it represents a druggable target for the identification of new agents for fighting antimicrobial resistance. Herein, we exploited tryptophan fluorescence spectroscopy (TFS) as a suitable technique for the identification of potential LsrK ligands from an in-house library of chemicals comprising synthetic compounds as well as secondary metabolites. Three secondary metabolites (Hib-ester, Hib-carbaldehyde and (R)-ASME) showed effective binding to LsrK, with KD values in the sub-micromolar range. The conformational changes were confirmed via circular dichroism and molecular docking results further validated the findings and displayed the specific mode of interaction. The activity of the identified compounds on the biofilm formation by some Staphylococcus spp. was investigated. Hib-carbaldehyde and (R)-ASME were able to reduce the production of biofilm, with (R)-ASME resulting in the most effective compound with an EC50 of 14 mg/well. The successful application of TFS highlights its usefulness in searching for promising LsrK inhibitor candidates with inhibitor efficacy against biofilm formation.

Novel S1R agonists counteracting NMDA excitotoxicity and oxidative stress: A step forward in the discovery of neuroprotective agents.

Sigma-1 receptor (S1R) has been considered a promising therapeutic target for several neurodegenerative diseases and S1R agonists have shown neuroprotective activity against glutamate excitotoxicity and oxidative stress. Starting from a previously identified low nanomolar S1R agonist, in this work we prepared and tested novel benzylpiperidine/benzylpiperazine-based compounds designed by applying a ring opening strategy. Among them, 4-benzyl-1-(2-phenoxyethyl)piperidine 6b (S1R Ki = 0.93 nM) and 4-benzyl-1-(3-phenoxypropyl)piperidine 8b (S1R Ki = 1.1 nM) emerged as high affinity S1R ligands and showed selectivity over S2R and N-methyl-d-aspartate receptor (NMDAR). Candidate compounds behaved as potent S1R agonists being able to enhance the neurite outgrowth induced by nerve growth factor (NGF) in PC12 cell lines. In SH-SY5Y neuroblastoma cell lines they exhibited a neuroprotective effect against rotenone- and NMDA-mediated toxic insults. The neuroprotective activity of 6b and 8b was reverted by co-treatment with an S1R antagonist, PB212. Compounds 6b and 8b were tested for cytotoxicity in-vitro against three human cancer cell lines (A549, LoVo and Panc-1) and in-vivo zebrafish model, resulting in a good efficacy/safety profile, comparable or superior to the reference drug memantine. Overall, these results encourage further preclinical investigations of 6b and 8b on in-vivo models of neurodegenerative diseases.

Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth.

Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

From Nature to Synthetic Compounds: Novel 1(N),2,3 Trisubstituted-5-oxopyrrolidines Targeting Multiple Myeloma Cells.

The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Urea-based anticancer agents. Exploring 100-years of research with an eye to the future.

Suramin was the first urea-based drug to be approved in clinic, and in the following century a number of milestone drugs based on this scaffold were developed. Indeed, urea soon became a privileged scaffold in medicinal chemistry for its capability to establish a peculiar network of drug-target interactions, for its physicochemical properties that are useful for tuning the druggability of the new chemical entities, and for its structural and synthetic versatility that opened the door to numerous drug design possibilities. In this review, we highlight the relevance of the urea moiety in the medicinal chemistry scenario of anticancer drugs with a special focus on the kinase inhibitors for which this scaffold represented and still represents a pivotal pharmacophoric feature. A general outlook on the approved drugs, recent patents, and current research in this field is herein provided, and the role of the urea moiety in the drug discovery process is discussed form a medicinal chemistry standpoint. We believe that the present review can benefit both academia and pharmaceutical companies' medicinal chemists to prompt research towards new urea derivatives as anticancer agents.

Effects of endocrine active contaminating pesticides on RACK1 expression and immunological consequences in THP-1 cells.

We have previously demonstrated that RACK1, which expression is under steroid hormone control, plays an important role in the activation of immune cells and its expression can be useful to evaluate the immunotoxic profile of endocrine disrupting chemicals (EDCs). Hence, we investigated the effects of three contaminating and persistent pesticides: the fungicide vinclozolin (VIN), the herbicide atrazine (ATR) and the insecticide cypermethrin (CYP) on RACK1 expression and on innate immune response. VIN resulted in modest alteration of RACK1 while ATR and CYP reduced in a dose dependent manner RACK1 expression, ultimately leading to the decrease in lipopolysaccharide-induced IL-8 and TNF-α release and CD86 and CD54 surface marker expression. Moreover, our data indicate that, after exposure to EDCs, alterations of RACK1 expression can also occur with mechanisms not directly mediated by an interaction with a nuclear or membrane steroid receptors. Therefore, RACK1 could represent a useful EDCs screening tool to evaluate their immunotoxic potential and to dissect their mechanisms of action.

Effects of endocrine disrupting chemicals on the expression of RACK1 and LPS-induced THP-1 cell activation.

The existence of a complex hormonal balance among glucocorticoids, androgens and estrogens involved in the regulation of Receptor for Activated C Kinase 1 (RACK1) expression and its related immune cells activation, highlights the possibility to employ this protein as screening tool for the evaluation of the immunotoxic profile of endocrine disrupting chemicals (EDCs), hormone-active substances capable of interfering with the physiologic hormonal signaling. Hence, the aim of this work was to investigate the effect of the exposure of EDCS 17α-ethynylestradiol (EE), diethyl phthalate (DEP) and perfluorooctanesulfonic acid (PFOS) on RACK1 expression and on lipopolysaccharide (LPS)-induced activation of the human monocytic cell line THP-1, a validated model for this investigation. In line with our previous results with estrogen-active compounds, EE treatment significantly induced RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which paralleled an increase in LPS-induced IL-8, TNF-α production and CD86 expression, previously demonstrated to be dependent on RACK1/PKCß activation. EE mediates its effect on RACK1 expression through G-protein-coupled estrogen receptor (GPER) and androgen receptor (AR) ligand-independent cascade, as also suggested by in silico molecular docking simulation. Conversely, DEP and PFOS induced a dose-dependent downregulation of RACK1 promoter transcriptional activity, mRNA expression, and protein levels, which was mirrored by a reduction of IL-8, TNF-α production and CD86 expression. Mifepristone pre-treatments abolish DEP and PFOS effects, confirming their GR agonist profile, also corroborated by molecular docking. Altogether, our data confirm that RACK1 represents an interesting target of steroid active compounds, which expression offers the opportunity to screen the immunotoxic potential of different hormone-active substances of concerns due to their human exposure and environmental persistence.