Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy-to-drug target is the coronavirus Main Protease (3CL^pro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.     

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2**

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.     

Application of a Fluorescence Anisotropy-Based Assay to Quantify Transglutaminase 2 Activity in Cell Lysates

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.     

重组大肠杆菌表达双功能谷胱甘肽合成酶的发酵工艺研究

对重组表达双功能谷胱甘肽合成酶（GshF）的大肠杆菌进行了5L罐的发酵工艺探索。分别考察了不同培养基、诱导剂种类、诱导时机、诱导温度、补料流加碳源类型等对菌体生长、GshF蛋白表达及其酶活的影响。结果表明,采用葡萄糖进行补料流加．在优化工艺条件下,GshF表达量达到1．94g·L^-1。