The persistent evolution of SARS-CoV-2 underscores the need for antiviral strategies. The viral main protease (Mpro) represents a conspicuous target due to its essential role in viral replication and high conservation across SARS-CoV-2. Conventional Mpro inhibitors face challenges such as drug resistance and toxicity. Proteolysis-targeting chimeras (PROTACs) offer an event-driven mechanism to degrade rather than inhibit the target protein, overcoming key limitations of occupancy-driven pharmacology. Here, we designed a series of thalidomide-based PROTACs targeting SARS-CoV-2 Mpro and explored their effectiveness through computational simulations and experimental validation. Molecular docking revealed that PROTACs A, B, and C exhibit favorable binding free energies (ΔG < -8.0 kcal mol-1). These findings were further supported by molecular dynamics simulations, which demonstrated consistently stable binding over 10 ns, with backbone RMSD values maintained within the range of 0.18-0.30 Å. Cell experiments indicated that PROTACs A, B, and C effectively induced dose-dependent Mpro degradation in HEK293 stable cells, with DC50 values ranging from 0.530 to 0.985 μM and exhibited high selectivity indices (CC50/DC50 > 10). Mechanistically, PROTACs-induced degradation of Mpro via the ubiquitin-proteasome system was evidenced by enhanced K48-linked polyubiquitination and suppression of degradation upon proteasome inhibition. The PROTACs (A, B and C) exhibit comparable effects and share similar mechanisms in degrading Mpro. Our work develops effective degraders targeting SARS-CoV-2 Mpro and highlights the therapeutic potential of PROTACs in combating drug-resistant viral targets via a catalytic degradation mechanism.

3CL protease (3CLpro), a key enzyme of SARS-CoV-2 replication, is one of the most selective targets of antivirals, as no homologous protease has been recognized in the human body. As proteolysis-targeting chimeras (PROTACs) are superior to traditional inhibitors, based on the reported cereblon (CRBN) ligands thalidomide and lenalidomide, 3CLpro ligands of peptidomimetic inhibitors, and suitable linkers, we aimed to develop novel PROTACs that may trigger efficient intracellular 3CLpro degradation through a balance of hydrophilicity and lipophilicity. In brief, we designed and synthesized 5 PROTAC molecules. The 3CLpro degradation efficiency of the PROTACs was assayed in stable SARS-CoV-2 3CLpro expression HEK293 cell models and evaluated by Western blot. All compounds showed prominent 3CLpro degradation activity with tolerable HEK293 cytotoxicity. The most prominent PROTAC compounds, 15 and 16, have DC50 values of approximately 1 µM, and Dmax of 89.3% and 75% respectively, indicating good potential for further application.

Male infertility is a global issue caused by poor sperm quality, particularly motility. Enhancement of the sperm quality may improve the fertilization rate in assisted reproductive technology (ART) treatment. Scriptaid, with a novel human sperm motility-stimulating activity, has been investigated as a prospective agent for improving sperm quality and fertilization rate in ART. We evaluated the effects of Scriptaid on asthenozoospermic (AZS) semen, including its impact on motility stimulation and protective effects on cryopreservation and duration of motility, by computer-aided sperm analysis (CASA). Sperm quality improvement by Scriptaid was characterized by increased hyaluronan-binding activity, tyrosine phosphorylation, adenosine triphosphate (ATP) concentration, mitochondrial membrane potential, and an ameliorated AZS fertilization rate in clinical intracytoplasmic sperm injection (ICSI) experiments. Furthermore, our identification of active Scriptaid analogs and different metabolites induced by Scriptaid in spermatozoa lays a solid foundation for the future biomechanical exploration of sperm function. In summary, Scriptaid is a potential candidate for the treatment of male infertility in vitro as it improves sperm quality, prolongs sperm viability, and increases the fertilization rate.

In order to make the synthetic route of Thiamidol more efficient and easier to scale up,a new method was developed.The compound 2,4-dihydroxyacetophenone was dissolved in ethyl acetate and brominated with cupric bromide.After bromination,the mixture was filtered to remove cuprous bromide.The obtained ethyl acetate solution containing 2-bromo-1-(2,4-dihydroxyphenyl)ethanone was directly reacted with(2-methylpropanoyl)thiourea to obtain the precipitate of Thiamidol hydrobromide.The precipitate was neutralized by deacidification and recrystallized to afford Thiamidol with high purity.In addition,in the screening of fertility-promoting substances,Thiamidol was found to be effective in protecting human sperm from motility loss in vitro.This suggests that it may have potential application in fertility protection and promotion.

Phosphodiesterase (PDE) inhibitors can improve sperm motility in patients with asthenozoospermia. However, the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity. We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil. After seminal plasma was discarded, several semen samples were subjected to four treatments (control, PF-2545920, pentoxifylline, and sildenafil) to evaluate their ability to affect motility, viability, and spontaneous acrosome reactions. Intracellular calcium and adenosine triphosphate (ATP), mitochondrial membrane potential, and penetration through viscous medium were assessed by flow cytometry, luciferase, and hyaluronic acid after treatment with PF-2545920. Statistical analyses were performed using the analysis of variance statistical test. PF-2545920 elevated the percentage of motile spermatozoa compared to the control, pentoxifylline, and sildenafil groups at 10 µmol l -1 ( P < 0.01). It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions ( P < 0.05). PF-2545920 also increased mitochondrial membrane potential ( P < 0.001) and altered intracellular calcium ( P < 0.05) in a dose-dependent manner, including increasing sperm hyaluronic acid penetrating ability ( P < 0.05). Therefore, PF-2545920 might be an excellent choice for stimulating the sperm motility.

Oncrasin-1类衍生物对来自肺癌、结肠癌等多种癌细胞系都具有高度活性,其中NSC-743380活性最佳.然而,它在结构上存在问题,易形成二聚体而失活,限制了进一步的研究发展.因此,本文以Oncrasin-1为先导化合物,设计了13种衍生物,通过引入氨基和酰胺基,尝试提高化合物稳定性并保留活性.化合物结构经1H NMR,13C NMR,HR-MS(ESI)表征,并通过细胞增殖实验(CCK-8实验)测定其对人胰腺癌细胞(PANC-1)、人宫颈癌细胞(HeLa)和人肝癌细胞(HepG2)的抗肿瘤活性.结果表明,13个Oncrasin-1类衍生物中,有10个表现出更好的活性,其中化合物4((4-(((1-(4-氯苄基)-1H-吲哚-3-基)甲基)氨基)丁基)氨基甲酸叔丁酯)对HeLa的IC50达到了3.98μM.

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid β-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

The 3C-like protease (also known as M-pro) plays a key role in SARS-CoV-2 replication and has similar substrates across mutant coronaviruses, making it an ideal drug target. We synthesized 19 thiazolidinedione derivatives via the Knoevenagel condensations and Mitsunobu reactions as potential 3C-like protease inhibitors. The activity of these inhibitors is screened in vitro by employing the enzymatic screening model of 3C-like protease using fluorescence resonance energy transfer. Dithiothreitol is included in the enzymatic reaction system to avoid non-specific enzymatic inhibition. Active inhibitors with diverse activity are found in this series of compounds, and two representative inhibitors with potent inhibitory activity are highlighted.

Endoplasmic reticulum membrane protein complex subunit 10 (EMC10) is an evolutionarily conserved and multifunctional factor across species. We previously reported that Emc10 knockout (KO) leads to mouse male infertility. Emc10-null spermatozoa exhibit multiple aspects of dysfunction, including reduced sperm motility. Two subunits of a Na/K-ATPase, ATP1A4 and ATP1B3, are nearly absent in Emc10 KO spermatozoa. Here, two isoforms of EMC10 were characterized in the mouse testis and epididymis: the membrane-bound (mEMC10) and secreted (scEMC10) isoforms. We present evidence that mEMC10, rather than scEMC10, is required for cytoplasm sodium homeostasis by positively regulating ATP1B3 expression in germ cells. Intra-testis mEMC10 overexpression rescued the sperm motility defect caused by Emc10 KO, while exogenous recombinant scEMC10 protein could not improve the motility of spermatozoa from either Emc10 KO mouse or asthenospermic subjects. Clinically, there is a positive association between ATP1B3 and EMC10 protein levels in human spermatozoa, whereas no correlation was proven between seminal plasma scEMC10 levels and sperm motility. These results highlight the important role of the membrane-bound EMC10 isoform in maintaining cytoplasm sodium homeostasis and sperm motility. Based on the present results, the mEMC10-Na, K/ATPase α4β3 axis is proposed as a novel mechanism underlying the regulation of cytoplasmic sodium and sperm motility, and its components seem to have therapeutic potential for asthenospermia.

Tideglusib is considered to be a promising alternative to nonyl alcohol-9 contraceptives. Previous studies have demonstrated that the rapid spermicidal effect of tideglusib at a high concentration (≥ 10 μM) may occur through detergent-like activity; however, the effect of low concentrations of tideglusib (< 5 μM) on sperm is unknown. We explored the intracellular mechanism of tideglusib (< 5 μM) on the immobilization of human sperm by exploring related signaling pathways in human sperm. After treatment with tideglusib (1.25 μM) for 2 h, sperm motility rate decreased to 0, while sperm membrane integrity rate was 70

合成了3个含有荧光基团的1,2,4-噻二唑-3,5-二酮衍生物,其结构经1H NMR,13C NMR,HR-MS(ESI)表征,并考察了这些衍生物对精子的制动活性和荧光标记作用.结果表明:与先导化合物Tideglusib相比,目标化合物均保持了较强的杀精活性,且在紫外光激发下,具有相似的荧光标记模式.

Human β-defensins are an important family of innate host defense peptides with pleiotropic activities. Human β-defensin 36 (DEFB136) is a novel member of the β-defensin family which have not been characterized so far. In the present research, the DEFB136 peptide was expressed successfully and purified using the IMPACT-TWIN 1 expression system. The purified DEFB136 peptide was identified by MALDI-TOF mass spectrometry and circular dichroism spectroscopy. While the recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans strains, but had low cytotoxicity to human erythrocytes. In addition, the result of the octet assay showed that the DEFB136 had a high lipopolysaccharide (LPS)-binding affinity, suggesting the DEFB136 may be involved in immunoregulation through its LPS neutralization. These results may help lay the groundwork to understand better the complex interaction between innate host defense and the diversity of the defensin family.

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H 2 S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

精液的凝固与液化是一种保护精子运动和正常生育能力的自然生理现象,为哺乳类和灵长类动物所特有.在正常成年男性的一次射精量中,精子只占7％左右,其余均为精浆,其中70％来自精囊,20％来自前列腺,剩下的3％来自尿道球腺[1].在混合各腺体部分后精液自发凝固呈粘稠胶冻状,使其射入阴道后不至于立即流失.凝固物在随后几分钟内液化,使活动精子逐渐释放.液化时间是评价精子质量的重要标准之一,室温下,正常精液标本在射精后60min内液化,且通常在15min内完成.如果超过60min仍不能液化或液化不完全则称为精液液化异常(abnormal semen liquefaction)[2],包括液化迟缓和不液化,是导致男性不育的重要原因,值得生殖医学界的重视与关注.

The family of human β-defensins consists of small cysteine-rich peptides, which are receiving significant attention due to their antimicrobial activity. The N-terminal cysteine motif of β-defensin is considered to contribute to its biological activity. Human β-defensin 118 (DEFB 118) is a particular anion β-defensin expressed predominantly in the male reproductive tract, but its physiological activity has not yet been revealed. In order to verify the potential role of the N-terminal domain of DEFB118 peptide in the regulation of infection, the truncated β-defensin core region of DEFB118 peptide was expressed with IMPACT-pTWIN1 system in Escherichia coli. Herein, the purified homogeneous DEFB118 peptide was identified by mass spectrometry and circular dichroism spectroscopy. The in vitro experiments revealed that DEFB118 peptide exhibited prominent LPS-binding potency (KD: 2.94 nM). Moreover, the DEFB118 core peptide significantly inhibited the mRNA level of LPS-induced inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in RAW264.7 cells, and correspondingly decreased secretion of IL-6 and TNF-α. We concluded that strong binding of DEFB118 to LPS might prevent LPS from binding to its receptor, and hence inhibited cytokines secretion. The results of this study may be a benefit to elucidate the immune protection of DEFB118 in the male reproductive tract.

Objective::Amino acyl modification of lysine residues is an essential mechanism of nutrient sensing that regulates various biological functions including reproduction. At present, the lack of pan-specific antibodies for a recently identified lysine valylation hinders the characterization and detection of this modification. The objective of this study is to raise pan-specific antibodies that may facilitate the identification of novel expression patterns of lysine valylation.Methods::Chicken ovalbumin was valylated as an immunogen to raise polyclonal antibodies (PcAbs) in rabbits. The population of the pan-specific antibodies recognizing valylated lysine was purified using the chemically synthesized valylated peptides consisting of random amino acids. The specificity of the antibodies was evaluated using ELISA, dot blots, Western blots, and immunohistochemistry (IHC) staining in human epididymis as well.Results::A preliminary and simple strategy to make an anti-valylated lysine PcAb was developed. The recognition of the antibodies to valyllysine was evaluated as pan specific. This was useful for the detection of the newly identified valyl modification using ELISA, dot blots, and Western blots. The antibodies were also successfully utilized in IHC assays, which revealed novel valyllysine modification patterns in epididymis tissues of human.Conclusions::A new antibody tool was provided for the study of lysine valylation. The novel expression patterns of valyllysine in the epididymis suggest that this modification may be involved in sperm maturation.

ObjectivesWe aimed to evaluate the antifertility activity and vaginal irritation effects of tideglusib in vivo using rabbit models and to evaluate the cytotoxical effects of tideglusib to sperm, vaginal cells and vaginal bacteria (L. acidophilus) in vitro.Study designWe treated female rabbits with vaginal tideglusib 1 mM, nonoxynol-9 (N-9) or vehicle control (Poloxamer 407). In experiment 1, we sacrificed females (n = 6 each) after 10 days of daily administration and assessed vaginal histological changes using Eckstein irritation score. In experiment 2, females (n = 9 each) received estradiol benzoate to induce ovulation 24 h prior to vaginal treatment followed by introduction of a fertile male. These females underwent necropsy at the 21st day to assess pregnancy status. In experiment 3, we used an HTM-TOX IVOS sperm motility analyzer and scanning electron microscopy (SEM) to evaluate the effect of tideglusib on human sperm samples. In experiment 4, we evaluated the effect of tideglusib on lactobacillus and vaginal cell growth in vitro.ResultsThe total irritation score of tideglusib vs. N-9 was 3.4 ± 2.07 vs. 7.8 ± 3.82, p<.05. The pregnancy rate of tideglusib, N-9 and control group was 11.1%, 0% and 88.9%, respectively. Tideglusib exhibited a dose-dependent spermostatic/spermicidal activity, and the minimum effective concentrations of tideglusib and N-9 were 8.724 ± 3.047 μM and 219.75 ± 41.78 μM, respectively. SEM and transmission electron microscopy revealed acrosomal membrane impairments caused by tideglusib. Tideglusib was much less toxic to vaginal cells and L. acidophilus than N-9 in vitro.ConclusionsEvaluation using rabbit models indicated that tideglusib is a prospective spermicidal contraceptive with low vaginal irritation effects.ImplicationsTideglusib or tideglusib analogues may be a contraceptive with perspective to replace N-9. It is possible for a spermicide to balance spermicidal activity and vaginal/cervical irritation effects very well.

Minyan Liu1, Zhiting Chen2, Yunxia Deng1, Yunlong Zhang1, Yiting Yang2, Yuhua Li2, Heguo Yu2, Hua Diao2* and Zhiyu Shao1* Author Affiliations 1College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China 2NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Basic Medical Sciences, Fudan University, Shanghai, China Received: November 06, 2019 | Published: November 26, 2019 Corresponding author: Hua Diao, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China Zhiyu Shao, NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Basic Medical Sciences, Fudan University, Shanghai, China DOI: 10.26717/BJSTR.2019.23.003875

Objective: SPAG11E is the first β-defensin that has been reported to activate Ca2+ uptake and sperm motility. However, the exact subcellular localization and interaction of SPAG11E with sperm remain controversial because of the lack of qualified antibody tools. SPAG11E is also a potential male antifertility target because SPAG11E fragment conjugated with a carrier protein exhibits male contraceptive vaccine potential. However, the fine B-cell epitope motifs of SPAG11E have not been analyzed, which hampered further exploration of the potential target. Methods: Polyclonal and monoclonal antibodies (mcAbs) of mature SPAG11E were raised and qualified with Western blotting. Subcellular localization of SPAG11E was revealed by Western blotting, immunohistochemistry staining, and electron microscopy. B-cell epitopes of rat SPAG11E were mapped by Western blotting using polyclonal and mcAbs. Based on the conservation of the identified epitope motifs between rat and mouse SPAG11E, antifertility potential of the epitope motifs was evaluated by the offspring of the males compromised with specific mcAbs. Results: SPAG11E antibodies of high quality were obtained and all B-cell epitope motifs of rat SPAG11E were mapped, in which conserved epitope motifs of SPAG11E in various species were discovered. The epitope motifs recognized by mcAbs were identified respectively. With mcAbs, rat SPAG11E was proved to be expressed in the caput region of epididymis. A novel finding was that SPAG11E was located in the flagella and nuclei of sperm as revealed by immunoelectron microscopy. In addition, the males treated with mcAbs (3#-1 and 10#B4) showed apparently fewer offspring. Conclusions: SPAG11E revealed a β-defensin with novel localization in sperm flagellum and nucleus with qualified antibodies. All B-cell epitope motifs of rat SPAG11E were determined, and the antifertility potential was proved by corresponding mcAbs.

Male contraceptive methods can be divided into barrier methods,pharmaceutical intervention methods and the other.Barrier methods include condoms,vasoligation,reversible inhibition sperm under guidance (RISUG) and Vasalgel,etc.Pharmaceutical intervention methods can be further classified as hormonal and non-hormonal male contraceptive.Hormone-based male contraceptives based on negative feedback regulation of the hypothalamic-pituitary-gonadal axis affect spermatogenesis for contraceptive purposes.Development of non-hormonal male contraceptive is a trend because of the advantages of high specificity,hypotoxicity and reversibility.In addition,there are some contraceptive researches by the use of radiation,microwaves,nanomaterials,and high-temperature sealing.Herein we focus on a systematic review of barrier methods and pharmaceutical intervention methods.