CD4 T cells are essential for protective immunity against Chlamydia in the female reproductive tract (FRT), yet the characteristics of protective mucosal effector CD4 T cells remain poorly defined. We previously identified the transcription factor BHLHE40 as a key regulator of polyfunctional effector CD4 T cell differentiation during Chlamydia infection. Here, we identify the chemokine receptor CXCR6 as a marker of these protective T cells. Following intravaginal Chlamydia muridarum infection, Bhlhe40-deficient mice exhibited reduced frequencies of CXCR6+ CD4 T cells that correlated with impaired bacterial control. CXCR6 expression on T cells was associated with loss of stem-like features and acquisition of an effector phenotype. Compared with CXCR6- cells, CXCR6+ CD4 T cells displayed enhanced proliferation and polyfunctionality by co-producing cytokines IFN-γ, IL-17A, and GM-CSF. Although CXCR6 was dispensable for CD4 T cell homing to the FRT, it promoted localization to the infected epithelium and the emerging memory lymphoid clusters. Importantly, depletion of CXCR6+ CD4 T cells reduced polyfunctional effectors and impaired bacterial clearance. Collectively, these findings identify CXCR6 as a marker of protective polyfunctional CD4 T cells and implicate CXCR6-dependent tissue positioning as a key component of effective mucosal immunity, highlighting CXCR6 as a potential biomarker for Chlamydia vaccine development.

Lung alveolar macrophages (AMs) originate from yolk sac and fetal liver monocytes during embryogenesis. AMs function as sentinels to airway pathogens and can be replaced by the infiltration of monocyte-derived-AMs (Mo-AMs) during inflammation. During Mycobacterium tuberculosis (Mtb) infection, AMs act as an early permissive niche to promote Mtb growth. However, the fate of AMs during long-term Mtb infection and how ontogeny impacts the permissiveness of AMs to Mtb remains to be elucidated. Combining single-cell RNA-seq with a fate-mapping approach, we revealed that long-term Mtb infection results in replenishment of embryonic-derived-AMs by Mo-AMs with a higher proinflammatory status, which implicates an enhanced capacity to control Mtb infection in those cells. To investigate the impact of ontogeny on AM responses to Mtb, we utilized a β-glucan intranasal inoculation model in which β-glucan induces highly glycolytic, phagocytic and proinflammatory Mo-AMs in the airway. Moreover, using single cell ATAC-seq and chemical inhibition, we demonstrated the requirement of the SWItch/Sucrose Non-Fermentable complexes in regulating inflammatory responses in Mo-AMs upon stimulation. Lastly, β-glucan-induced Mo-AMs produced higher levels of protective cytokines and limited pulmonary Mtb burden in mice. Collectively, these findings assert that monocytes contribute to the AM pool during Mtb infection and implicate ontogeny as an intrinsic determinant for AM responses to Mtb infection. Supported by NIH/NIAID R01AI184960; R21AI175738. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Azole fungicides disrupt pregnancy, but their effects on placental 3β-hydroxysteroid dehydrogenases (3β-HSDs) and structure-activity relationships (SARs) remain unclear. This study evaluated 9 azole fungicides for their inhibitory potency, mechanism, SAR, and docking interactions with human 3β-HSD1 and rat 3β-HSD4. Bromuconazole, climbazole, cyproconazole, and etaconazole inhibited human 3β-HSD1 (IC50 = 6.21 μM to 104.83 μM), with climbazole being the most potent. These compounds acted as mixed/noncompetitive inhibitors and climbazole suppressed progesterone secretion in human JAr cells at ≥ 2.5 μM. They also inhibited rat 3β-HSD4, albeit with lower potency. Molecular docking revealed that these fungicides bind to the NAD+/steroid-binding site via hydrogen bonds, hydrophobic interactions, and van der Waals forces. A negative correlation between LogP and IC50 indicated that lipophilicity enhances inhibitory efficacy. 3D-QSAR analysis further confirmed the critical role of hydrogen bonding in binding affinity. In conclusion, climbazole exhibits the strongest inhibition of human 3β-HSD1, driven by its high lipophilicity. These findings highlight the endocrine-disrupting potential of azole fungicides and provide insights into their SAR and inhibition mechanisms, aiding risk assessment and regulatory decisions.

Cannabidiol (CBD), a major cannabinoid found in Cannabis sativa L., has been used in the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex. Recently, concerns have been raised regarding the male reproductive toxicity of CBD in animal models, such as monkeys, rats, and mice. In our previous studies, we reported that CBD inhibited cell proliferation in both primary human Sertoli cells and mouse Sertoli TM4 cells. Transcriptomic analysis revealed that in primary human Sertoli cells CBD disrupted DNA replication, cell cycle, and DNA repair, ultimately causing cellular senescence. In this study, we further investigated the molecular changes induced by CBD in mouse Sertoli TM4 cells using RNA-sequencing analyses and compared the transcriptomic profile with that of primary human Sertoli cells. Our findings demonstrated that, unlike in primary human Sertoli cells, CBD did not induce cellular senescence but caused apoptosis in mouse Sertoli TM4 cells. Through transcriptomic data analysis in mouse Sertoli TM4 cells, immune and cellular stress responses were identified. Moreover, transcriptomic comparisons revealed major differences in molecular changes induced by CBD between mouse Sertoli TM4 and primary human Sertoli cells. This suggests that primary human Sertoli cells and mouse Sertoli cells may respond differently to CBD.

Studies of the planar cell polarity (PCP) protein complexes Fat1/Fjx1 and Dchs/Fjx1 that form heterotypic interacting bridges of Fat1-Dchs between adjacent cells to confer PCP, as noted in Drosophila, are also found in mammalian cells and tissues as orthologs, such as in Sertoli cells and condensed spermatids in the seminiferous epithelium of the testis. Recent studies have shown that these two interacting PCP protein complexes are also crucial regulators of microtubule and actin dynamics, modulating the polymerization of both microtubules and actin filaments in the testis. In this review, we provide a brief update and thought-provoking concept on the PCP core proteins and the associated downstream signaling pathways utilized by PCP proteins to confer PCP and regulation of the microtubule and actin cytoskeletons in the testis. However, we focus on recent data in the field on the Fat1/Fjx1 and Dchs/Fjx1 protein complexes, which are also heterotypic interacting protein complexes, and their functional role in modulating the microtubule and actin cytoskeletal organization. Based on these recent findings, we formulate a hypothetic model depicting the role of these two PCP protein complexes in modulating the timely "opening" and "closing" of the blood-testis barrier (BTB) formed by adjacent Sertoli cells near the base of the seminiferous epithelium. Additionally, these two PCP protein complexes also modulate cytoskeletal dynamics between Sertoli cells and condensed spermatids to support haploid spermatid transport across the seminiferous epithelium during their structural transformation through spermiogenesis, and their eventual release at spermiation during the epithelial cycle of spermatogenesis. This hypothetical model will provide a useful framework for designing functional experiments to understand the role of PCP proteins in supporting spermatogenesis.

CD4 T cells are essential for host immunity to the intracellular bacterium Chlamydia, and the formation of tissue-resident memory CD4 T cells (CD4 TRM) in the female reproductive tract (FRT) is key for vaccine-induced protective immunity. As shown in previous studies, the pleiotropic cytokine transforming growth factor beta (TGFβ) regulates T cell migration, effector function, and memory maintenance in a context-dependent manner. To investigate the role of TGFβ signaling during Chlamydia infection in the FRT, we co-transferred WT and Tgfbr2fl/fl dLck-cre (Tgfbr2-/-) CD4 T cells into Tcrβ-/- recipient mice and compared CD4 T cell responses following Chlamydia muridarum intravaginal infection. At day 14, the ratio of WT:Tgfbr2-/- KO CD4 T cells was much higher in the FRT than in the draining iliac lymph nodes (DLNs), indicating that TGFβ signaling promotes CD4 T cell migration/homing to the FRT during the effector phase. A similar trend was observed at a memory time point (56 dpi), with reduced CD69 expression in the Tgfbr2-/- CD4 T cell compartment, suggesting that TGFβ signaling is essential for CD4 TRM maintenance in the FRT. The reduced effector responses and memory maintenance in Tgfbr2-/- CD4 T cells resulted in poor protective immunity, as Tgfbr2-/- mice exhibited higher bacterial burden than WT during secondary C. muridarum infection. Together, these data support the critical roles of TGFβ signaling in CD4 T cells during acute bacterial infection at the FRT mucosa. Mucosal and Regional Immunology (MUC)

Microtubule-associated proteins (MAPs) refer to a large superfamily of proteins that bind to microtubules (MTs) structurally, modulating the rapid transition of MTs from a stable state (polymerized) to shrinkage (or catastrophe) via depolymerization through a meta-stable state. Changes of MTs from an assembled structure as linear protofilaments that are a packed/bundled ultrastructure to disassembled subunits of heterodimers of α-/ß-tubulins (or oligomers) can take place in milliseconds within a living cell. These heterodimers can also be rapidly phosphorylated, becoming GTP-bound, or rapidly polymerized into linear protofilaments of MT again. It is such rapid cyclic changes of MTs that support cellular development, growth, and changes in cell shape in response to changes in development or other physiological phenomena, such as the series of cellular events during spermatogenesis, cell divisions, and in response to environmental toxicants to protect cellular life. In this review, we seek to give a concise update and discussion on MAPs. Particularly, we focus on a specific member of the structural MAPs, namely MAP1a, and its interaction with the microtubule affinity regulatory kinases (MARKs, including MARK1, 2, 3, and 4, all are Ser/Thr protein kinases) in particular MARK4, and how these two MAPs work together to regulate MT dynamics in Sertoli cells to support germ cell development. This information should be helpful to investigators who seek to better understand the role of MAPs in testis biology.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

The role of motor proteins in supporting intracellular transports of vesicles and organelles in mammalian cells has been known for decades. On the other hand, the function of motor proteins that support spermatogenesis is also well established since the deletion of motor protein genes leads to subfertility and/or infertility. Furthermore, mutations and genetic variations of motor protein genes affect fertility in men, but also a wide range of developmental defects in humans including multiple organs besides the testis. In this review, we seek to provide a summary of microtubule and actin-dependent motor proteins based on earlier and recent findings in the field. Since these two cytoskeletons are polarized structures, different motor proteins are being used to transport cargoes to different ends of these cytoskeletons. However, their involvement in germ cell transport across the blood-testis barrier (BTB) and the epithelium of the seminiferous tubules remains relatively unknown. It is based on recent findings in the field, we have provided a hypothetical model by which motor proteins are being used to support germ cell transport across the BTB and the seminiferous epithelium during the epithelial cycle of spermatogenesis. In our discussion, we have highlighted the areas of research that deserve attention to bridge the gap of research in relating the function of motor proteins to spermatogenesis.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40 -/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40 -/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40 -deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia .

Abstract The sexually transmitted Chlamydia infection has been increasing in the last few decades in the United States and worldwide. A better understanding of CD4 T cell effector function during infection is essential to develop a protective vaccine. Recently, we reported that mice lacking the transcription factor basic helix-loop-helix family member e40 (BHLHE40) had a significant defect in their ability to clear infection from the female reproductive tract (FRT). An increase in IL-10-producing T regulatory type 1 (Tr1) cells and a decrease in polyfunctional CD4 T cells co-producing IFN-γ, IL-17A, and GM-CSF in Bhlhe40-/- mice correlated with greater bacterial burden and reduced protective immunity in these mice. Using scRNA-seq, we found that Cxcr6, which encodes the chemokine receptor CXCR6, was enriched in the polyfunctional CD4 T cell clusters in WT mice but not in the stem-like clusters in Bhlhe40-/- mice. Here, we found that both CXCR6- and CXCR6+ CD4 T cells were detected in the FRT following infection, but CXCR6+ CD4 T cells contained most of the polyfunctional T cells. Notably, anti-CXCR6-treated mice exhibited a marked decrease in polyfunctional CD4 T cells, particularly, those capable of producing IL-17A. This corresponded to increased bacterial burden and delayed clearance following intravaginal infection. Overall, our results demonstrate that CXCR6 marks a polyfunctional CD4 T population required for protective immunity against Chlamydia in the FRT.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3β-HSD2 with IC50 values of 114.79, 106.98, and 5.40 μM, respectively. For pig 3β-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 μM, respectively. Similarly, for rat 3β-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 μM, respectively. They were mixed inhibitors of pig and rat 3β-HSD, while triphenyltin was identified as a competitive inhibitor of human 3β-HSD2. The mechanism underlying the inhibition of organotins on 3β-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3β-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

BACKGROUND: CD8 + T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 (purinergic receptor P2X7) receptor to CD8T activation and subsequent promotion of sodium retention in the kidney. METHODS: We used mouse models of hypertension. Wild type were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-knockout mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously and kidneys were obtained at different experimental end points. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in cocultures of CD8Ts and mouse DCTs. RESULTS: We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to deoxycorticosterone acetate/salt treatment, similar to wild-type mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension. CONCLUSIONS: Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T-cell-mediated immunopathology in hypertension.

Curcuminoids have many pharmacological effects. They or their metabolites may have side effects by suppressing 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3). Herein, we investigated the inhibition of curcuminoids and their metabolites on human and rat 17β-HSD3 and analyzed their structure-activity relationship (SAR) and performed in silico docking. Curcuminoids and their metabolites ranked in terms of IC50 values against human 17β-HSD3 were bisdemethoxycurcumin (0.61 μM) > curcumin (8.63 μM) > demethoxycurcumin (9.59 μM) > tetrahydrocurcumin (22.04 μM) > cyclocurcumin (29.14 μM), and those against rat 17β-HSD3 were bisdemethoxycurcumin (3.94 μM) > demethoxycurcumin (4.98 μM) > curcumin (9.62 μM) > tetrahydrocurcumin (45.82 μM) > cyclocurcumin (143.5 μM). The aforementioned chemicals were mixed inhibitors for both enzymes. Molecular docking analysis revealed that they bind to the domain between the androstenedione and NADPH active sites of 17β-HSD3. Bivariate correlation analysis showed a positive correlation between LogP and pKa of curcumin derivatives with their IC50 values. Additionally, a 3D-QSAR analysis revealed that a pharmacophore model consisting of three hydrogen bond acceptor regions and one hydrogen bond donor region provided a better fit for bisdemethoxycurcumin compared to curcumin. In conclusion, curcuminoids and their metabolites possess the ability to inhibit androgen biosynthesis by directly targeting human and rat 17β-HSD3. The inhibitory strength of these compounds is influenced by their lipophilicity and ionization characteristics.

Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 and regulatory transcription factor Nr3c2 at 100–200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 μM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1, which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis.

The use of alternative substances to replace bisphenol A (BPA) has been encouraged. The objective of this study was to evaluate the effects of BPA and 9 BPA alternatives on human and rat aromatase (CYP19A1) in human and rat placental microsomes. The results revealed that bisphenol A, AP, B, C, E, F, FL, S, and Z, and 4,4 '-thiodiphenol (TDP) inhibited human CYP19A1 and bisphenol A, AP, B, C, FL, Z, and TDP inhibited rat CYP19A1. The IC50 values of human CYP19A1 ranged from 3.3 to 172.63 mu M and those of rat CYP19A1 ranged from 2.20 to over 100 mu M. BPA alternatives were mixed/competitive inhibitors and inhibited estradiol production in BeWo placental cells. Molecular docking analysis showed that BPA alternatives bind to the domain between heme and steroid and form a hydrogen bond with catalytic residue Met374. Pharmacophore analysis showed that there were one hydrogen bond donor, one hydrophobic region, and one ring aromatic hydrophobic region. Bivariate correlation analysis showed that molecular weight, alkyl atom weight, and LogP of BPA alternatives were inversely correlated with their IC50 values. In conclusion, BPA alternatives can inhibit human and rat CYP19A1 and the lipophilicity and the substituted alkyl size determines their inhibitory strength.

Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-gamma promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB.

Dithiocarbamates have been widely used in various industrial applications, such as insecticides (ferbam) or drug (disulfiram). This study explored the inhibitory effects of dithiocarbamates on human and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD) and investigated the structure-activity relationship and mechanistic insights. The inhibitory activity of six dithiocarbamates and thiourea on the conversion of pregnenolone to progesterone was evaluated using human KGN cell and rat testicular microsomes, with subsequent progesterone measurement using HPLC-MS/MS. The study found that among the tested compounds disulfiram, ferbam, and thiram exhibited significant inhibitory activity against human 3β-HSD2 and rat 3β-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3β-HSD2 and mixed/noncompetitive inhibition for rat 3β-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3β-HSD enzymes. The study also delved into the penetration of these dithiocarbamates through the human KGN cell membrane and their impact on progesterone production, highlighting their potency in inhibiting human 3β-HSD2. Furthermore, bivariate correlation analysis revealed a positive correlation of LogP (lipophilicity) with IC50 values for both enzymes. Docking analysis indicated that dithiocarbamates bind to NAD+ and steroid-binding sites, with some interactions with cysteine residues. In conclusion, this study provides valuable insights into the structure-activity relationship and mechanistic aspects of dithiocarbamates as inhibitors of human and rat gonadal 3β-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues.

Benzophenone chemicals (BPs) have been developed to prevent the adverse effects of UV radiation and they are widely contaminated. 11 beta-Hydroxysteroid dehydrogenase 1 (11 beta-HSD1) catalyze the conversion of inactive glucocorticoid to active glucocorticoid, playing critical role in many physiological function. However, the direct effect of BPs on human, pig, rat, and mouse 11 beta-HSD1 remains unclear. In this study, we screened the inhibitory strength of 12 BPs on 4 species, and performed the structure-activity relationship (SAR) and in silico docking analysis. The inhibitory potency of BPs was: for human 11 beta-HSD1, BP6 (IC50 = 18.76 mu M) > BP8 (40.84 mu M) > BP (88.89 mu M) > other BPs; for pig 11 beta-HSD1, BP8 (45.57 mu M) > BP6 (59.44 mu M) > BP2 (65.12 mu M) > BP (135.56 mu M) > other BPs; for rat 11 beta-HSD1, BP7 (67.17 mu M) > BP (68.83 mu M) > BP8 (133.04 mu M) > other BPs; and for mouse 11 beta-HSD1, BP8 (41.41 mu M) > BP (50.61 mu M) > other BPs. These BP chemicals were mixed/ competitive inhibitors of these 11 beta-HSD1 enzymes. The 2,2 '-dihydroxy substitutions in two benzene rings play a key role in enhancing the effectiveness of inhibiting 11 beta-HSD1, possibly via increasing hydrogen bond interactions. Docking analysis shows that these BPs bind to NADPH/glucocorticoid binding sites and forms hydrogen bonds with catalytic residues Ser and/or Tyr. In conclusion, this study demonstrates that BP chemicals can inhibit 11 beta-HSD1 from 4 species, and there are subtle species-dependent difference in the inhibitory strength and structural variations of BPs.