Androgens, such as 5α-dihydrotestosterone (5α-DHT), regulate numerous functions by binding to nuclear androgen receptors (ARs) and potential unknown membrane receptors. Here, we report that the androgen 5α-DHT activates membrane receptor GPR133 in muscle cells, thereby increasing intracellular cyclic AMP (cAMP) levels and enhancing muscle strength. Further cryoelectron microscopy (cryo-EM) structural analysis of GPR133-Gs in complex with 5α-DHT or its derivative methenolone (MET) reveals the structural basis for androgen recognition. Notably, the presence of the “Φ(F/L)2.64-F3.40-W6.53” and the “F7.42××N/D7.46” motifs, which recognize the hydrophobic steroid core and polar groups, respectively, are common in adhesion GPCRs (aGPCRs), suggesting that many aGPCRs may recognize different steroid hormones. Finally, we exploited in silico screening methods to identify a small molecule, AP503, which activates GPR133 and separates the beneficial muscle-strengthening effects from side effects mediated by AR. Thus, GPR133 represents an androgen membrane receptor that contributes to normal androgen physiology and has important therapeutic potentials.

Silyl ether is particularly attractive for application in drug development for its easy preparation, non-toxicity and remarkable biocompatibility. Earlier studies relied on the use of intracellular acidic conditions to induce the cleavage of alkoxy silyl ethers. However, acidic conditions are not suitable to trigger the release of phenoxy silyl ethers, since they are more stable under acidic conditions compared with neutral conditions. We explored the vulnerability of the phenoxy silyl ether towards biological nucleophilic reagents and found that glutathione (GSH) could effectively and selectively induce the cleavage of phenoxy silyl ether. We also demonstrated that the rate of cleavage was controllable by adjusting the substituents on the phenyl ring. Phenoxy silyl ether-based prodrugs and antibody-drug conjugates (ADCs) were designed and synthesized, which could be effectively activated in cells with high GSH levels and there was an obvious therapeutic window between cells with different GSH levels.

Antibody-drug conjugates (ADCs) have emerged as a highly promising modality for the treatment of various tumors, including pancreatic cancer. Due to the modular nature of ADCs, their efficacy is heavily influenced by the choice of antibody, payload, and linker. Given the therapeutic potential of triptolide for pancreatic cancer, this study aims to harness triptolide as the cytotoxic payload to construct ADCs targeting pancreatic cancer. Silyl ethers were utilized for the first time as cleavable linkers to connect triptolide with an antibody. This is because silyl ethers can be easily synthesized and the rate of drug release can be regulated by modifying the silyl ether groups. The release profile of the resulting linkers was investigated. And considering the balance between cleavage and stability, one silyl ether-based linker was selected to prepare an ADC, named A10. Meanwhile, a traditional dipeptide linker-based ADC, A9, was synthesized for comparison. The ADC A10 demonstrated superior inhibitory effects compared to ADC A9, both in vitro and in vivo. A10 displayed targeted cytotoxicity against cells with high PD-L1 expression and demonstrated a bystander killing effect on cells with low PD-L1 expression. In vivo imaging studies indicated that fluorescently labeled A10 accumulated in tumor regions. Additionally, significant antitumor activities of A10 were observed against Panc 08.13-derived tumor xenografts.

Although research on nitrosyl (NO) heme complexes and their one-electron reduced form, nitroxyl (or nitroxyl anion, NO-) derivatives, has been going on for decades, there are still disagreements about the electrical configuration of nitroxyl complexes, and the majority of the work on this topic is based on theoretical calculations. Following the initial nitroxyl iron porphyrin crystal structure, we present two further polymorphic forms of [CoCp2][Fe(TFPPBr8)(NO)]. Using the same completely halogenated porphyrin ligand, we also present two polymorphic forms of nitrosyl cobalt(II) complexes, which are another sort of {MNO}(8) structure. In addition to the EXANES and EPR studies of these {FeNO}(7) and {CoNO}(8) complexes, the {FeNO}(8) [CoCp2][Fe(TFPPBr8)(NO)] complex is also investigated by temperature-dependent Mossbauer experiments for the first time with the {FeNO}(7) precursor as a control sample. The analysis of the Mossbauer and crystal structural parameters between these two types of {MNO}(8) (M = Fe or Co) species and previously reported analogous ones allow us to conclude that the electronic configuration of [Fe(TFPPBr8)(NO)](-) is best described as an intermediate between low-spin Fe(II)-NO- and Fe(I)-NO center dot.

Phenyldivinylsulfonamides emerged from a series of divinylsulfonamides, demonstrating their ability to effectively re-bridge disulfide bonds. This kind of linkers was attached to monomethyl auristatin E (MMAE) and further conjugated with a model antibody, trastuzumab. After optimization, the linker 20 can deliver stable and highly homogenous DAR (Drug-to-Antibody Ratio) four antibody-drug conjugates (ADCs). The method was also applicable for other IgG1 antibodies to obtain ADCs with controlled four payloads. Moreover, the MMAE-bearing ADC is potent, selective and efficacious against target cell lines.

The lack of direct proof in either natural or synthetic systems for trans -dinitrosyl hemes, a key intermediate in the reactions of heme proteins (e.g. soluble guanylate cyclase (sGC), cytochrome c′ and So H-NOX) with nitric oxide (NO), has hampered understanding of the exact reaction mechanisms, such as the formation of the five-coordinate heme complex with NO at the proximal side (5c NO P ). Herein, we report the first isolation of a dinitrosyl metalloporphyrin complex, the six-coordinate, low-spin {Mn(NO) 2 } 7 species [Mn(TPP)(NO) 2 ] (TPP 2− = meso -tetraphenylporphyrin dianion). The complex shows distinct features, such as an elongated axial bond (1.877(9) vs. 1.641(5) Å), a higher NO stretching bond position (1760 vs. 1735 cm −1 ) and an isotropic resonance at g = 2.0, in sharp contrast to those of five-coordinate mononitrosyl analogues. In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and EPR studies provided deep insight into the reaction processes, demonstrating different responses of porphyrinates to NO.

Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii . Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6 th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.

Self-immolative linkers have been widely used to construct prodrugs to improve their efficacy and safety. In this study, we report the use of phenoxysilyl linker as a self-immolative unit to prepare antibody-drug conjugates (ADCs). Phenoxysily based ADC Ate-PPS-CA4 was prepared and its release was systematically investigated by mass spectrometry. Biological evaluation showed that Ate-PPS-CA4 displayed the ability to target delivery and self-immolative release the active payload CA4 on PD-L1 positive cells MDA-MB-231. As the same with its payload CA4, it could arrest the cell cycle to the G2/M phase and induced changes in cell morphology at the dose of its IC50. The development of this linker with novel drug release mechanisms will expand the methodology to construct ADCs, especially for non-internalizing ADCs by extracellular cleavage.

B7–H3 (immunoregulatory protein B7-homologue 3) is overexpressed in many cancer cells with limited expression in normal tissues, considered to be a promising target for tumor therapeutics. Clinical trials of antibody-drug conjugates (ADCs) against different targets for glioblastoma have been investigated and showed potent efficacies. In this study, we developed a homogeneous ADC 401-4 with a drug-to-antibody ratio (DAR) of 4, which was prepared by conjugation of Monomethyl auristatin E (MMAE) to a humanized anti-B7-H3 mAb 401, through a divinylsulfonamide-mediated disulfide re-bridging approach. In vitro studies, 401-4 displayed specific killing against B7–H3-expressing tumors and was more effective in cells with higher levels of B7–H3 for different glioblastoma cells. 401-4 was furthered labeled with Cy5.5 to yield a fluorescent conjugate 401-4-Cy5.5. The in vivo imaging studies showed that the conjugate accumulated in tumor regions and exhibited the ability to target-specific delivery. In addition, significant antitumor activities for 401-4 was observed against U87-derived tumor xenografts in a dose dependent manner.

Antibody-drug conjugates (ADCs) have emerging as efficient agents to target deliver cytotoxic drugs and reduce their off-target side effects. Triptolide has attracted attention to be used in ADC development. Herein, three rationally designed triptolide drug-linkers have been synthesized for use in site-specific construction of ADCs. Carbamates that were supposed to be more stable than carbonates were introduced to attach triptolide to the linkers. PEG and discrete PEG chains were incorporated to improve the hydrophilicity of drug-linkers. The ADCs were finally site-specifically prepared by conjugation of the drug-linkers to trastuzumab through disulfide re-bridging approach. The preliminary anti-tumor activities of these ADCs were evaluated and they displayed high potencies against HER2-targeted cancer in vitro and in vivo.

In the COVID-19 outbreak year 2020, a consensus was reached on the fact that SARS-CoV-2 spreads through aerosols. However, finding an efficient method to detect viruses in aerosols to monitor the risk of similar infections and enact effective control remains a great challenge. Our study aimed to build a swirling aerosol collection (SAC) device to collect viral particles in exhaled breath and subsequently detect SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR). Laboratory tests of the SAC device using aerosolized SARS-CoV-2 pseudovirus indicated that the SAC device can produce a positive result in only 10 s, with a collection distance to the source of 10 cm in a biosafety chamber, when the release rate of the pseudovirus source was 1,000,000 copies/h. Subsequent clinical trials of the device showed three positives and 14 negatives out of 27 patients in agreement with pharyngeal swabs, and 10 patients obtained opposite results, while no positive results were found in a healthy control group (n = 12). Based on standard curve calibration, several thousand viruses per minute were observed in the tested exhalations. Furthermore, referring to the average tidal volume data of adults, it was estimated that an exhaled SARS-CoV-2 concentration of approximately one copy/mL is detectable for COVID-19 patients. This study validates the original concept of breath detection of SARS-CoV-2 using SAC combined with RT-PCR.

We describe the use of natural product combretastatin A4 (CA4) as a versatile new payload for the construction of antibody-drug conjugates (ADCs). Cetuximab conjugates consisting of CA4 derivatives were site-specially prepared by disulfide re-bridging approach using cleavable and non-cleavable linkers. These ADCs retained antigen binding and internalization efficiency and exhibited high potencies against cancer cell lines in vitro. The conjugates also demonstrated significant antitumor activities in EGFR-positive xenograft models without observed toxicities. CA4 appears to be a viable payload option for ADCs research and development.

Disulfide re-bridging strategy has demonstrated significant advantages in the construction of homogeneous antibody drug conjugates (ADCs). However, a major issue that disulfide scrambling at the hinge region of antibody leads to the formation of "half-antibody" has appeared for many re-bridging linkers. We present bis(vinylsulfonyl)piperazines (BVP) as efficient linkers to selectively re-bridge disulfides at the antigen-binding fragment (Fab) regions and produce highly homogeneous conjugates with a loading of two drugs without disulfide scrambling. We also found that optically active (S)-configuration linkers led to more sufficient conjugation compared with (R)-configuration. The BVP-linked ADCs demonstrated superior efficacy and antigen-selectivity in vitro cytotoxicity.

We describe an efficient decision tree searching strategy (DTSS) to boost the identification of cross-linked peptides. The DTSS approach allows the identification of a wealth of complementary information to facilitate the construction of more protein-protein interaction networks for human cell lysate, which was tested by the use of a recently reported cross-linking data set (ACS Cent. Sci. 2019, 5, 1514-1522). A variant of the PhoX-linker, named pDSPE, was synthesized and applied to cross-link Escherichia coli cell lysate to demonstrate that the acquisition of doubly charged ions can significantly improve identification results. The method can be seamlessly integrated to other search engines to maximize the number of identified cross-links.

Methods that site-specifically attach payloads to an antibody with controlled DAR (Drug-Antibody Ratio) are highly desirable for the generation of homogeneous antibody-drug conjugates (ADCs). We describe the use of N-phenyl-divinylsulfonamide scaffold as a linker platform to site-specifically construct homogeneous DAR four ADCs through a disulfide re-bridging approach. Several monomethyl auristatin E (MMAE)-linkers were synthesized and the drug-linkers that contain electron-donating groups on the phenyl of the linker showed high stability. Her2-targeted MMAE-linker-herceptin and EGFR targeted MMAE-linker-cetuximab conjugates were prepared. The conjugates demonstrated high efficacy and selectivity for killing target-positive cancer cells in vitro. The EGFR-targeted conjugates also showed significant antitumor activities in vivo.

The title complex, [Fe(C 44 H 28 N 4 )(C 5 H 8 N 2 ) 2 ]·C 4 H 8 O, possesses inversion symmetry with the iron(II) atom located on a center of symmetry. The metal atom is coordinated in a symmetric octahedral geometry by four pyrrole N atoms of the porphyrin ligand in the equatorial plane and two N atoms of 1-ethylimidazole ligands in the axial sites; the complex crystallizes with a tetrahydrofuran solvent molecule. The average Fe—N p (N p is a porphyrin N atom) bond length is 1.995 (3) Å and the axial Fe—N Im (N Im is an imidazole N atom) bond length is 1.994 (2) Å. The two 1-ethylimidazole ligands are mutually parallel. The dihedral angle between the 1-ethylimidazole plane and the plane of the closest Fe—N p vector is 24.5°. In the crystal, the only significant intermolecular interactions present are C—H...π interactions.

Einfach und selektiv: Die Phosphorylierung von Tyrosin ist eine zentrale posttranslationale Modifikation, die die enzymatische Aktivität, Proteinkonformation und die Wechselwirkungen zwischen Proteinen reguliert. Über die hocheffiziente genetische Aufnahme von 3,5-Difluortyrosin in E. coli und die Anwendung von F2Y als 19F-NMR-Sonde für die Phosphorylierung von Tyrosin wird berichtet (siehe Bild).