Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

Development of Conformationally Restricted Negamycin Derivatives for Potent Readthrough Activity.

(+)-Negamycin, which is a dipeptide-like antibiotic containing a hydrazide structure, exhibits readthrough activity, resulting in the restoration of dystrophin in the mdx mouse model of Duchenne muscular dystrophy (DMD). In our previous structure-activity relationship study of negamycin, we found that its natural analogue 3-epi-deoxynegamycin (TCP-107), without antimicrobial activity, showed a higher readthrough activity than negamycin. In this study, we designed and synthesized cyclopropane-based conformationally restricted derivatives of TCP-107 and evaluated their readthrough activity in the cell-based reporter assay against a TGA-type mutation derived from DMD. As a result, a down-cis isomer, TCP-304, showed significant readthrough activity among the four isomers. Moreover, TCP-306, a derivative acylated by l-α-aminoundecanoic acid, possessed approximately 3 times higher activity than TCP-304. These down-cis derivatives showed dose-dependent readthrough activity and were effective for not only TGA but also TAG mutations. These results suggest that the conformational restriction of negamycin derivatives by the introduction of the cyclopropane ring is effective for an exhibition of potent readthrough activity.

[Long-Term Survival with Distal Pancreatectomy and Postoperative Chemotherapy in a Patient of Pancreatic Invasive Ductal Adenocarcinoma of the Tail with Peritoneal Dissemination].

A 68-year-old man was referred to our hospital for detailed examination of the pancreatic tail tumor. The tumor was diagnosed as the pancreatic invasive ductal adenocarcinoma and the distal pancreatectomy was scheduled. During surgery, a 2 mm white nodule was observed on the posterior wall of the stomach. Intraoperative frozen section showed no obvious malignant findings, suggesting leiomyoma or gastrointestinal stromal tumor. Distal pancreatectomy with D2 lymphadenectomy was completed as planned. However, this nodule was later confirmed by permanent pathological specimen to be peritoneal dissemination of pancreatic cancer and final diagnosis was invasive ductal carcinoma of pancreatic tail, pT3, pN1a, M1 (PER), pStage Ⅳ. He received chemotherapy for 17 months. Although liver metastasis was appeared 26 months after surgery, the disease is still being controlled with chemotherapy at 33 months.

New Negamycin-Based Potent Readthrough Derivative Effective against TGA-Type Nonsense Mutations.

We report a novel negamycin derivative TCP-1109 (13x) which serves as a potent readthrough drug candidate against nonsense-associated diseases. We previously demonstrated that TCP-112 (7), a nor-compound of native 3-epi-deoxynegmaycin, showed a higher readthrough activity than (+)-negamycin. In the present study, we performed a structure-activity relationship (SAR) study of compound 7 focused on its 3-amino group in an effort to develop a more potent readthrough compound. Introduction of a variety of natural or unnatural amino acids to the 3-amino group gave us the more potent derivative 13x which has about four times higher readthrough activity than 7 in a cell-based assay using a premature termination codon of TGA derived from Duchenne muscular dystrophy. The activity was dose-dependent and relatively selective for TGA. However, the activities for TAG and TAA were also higher than those of (+)-negamycin and 7. Moreover, compound 13x showed significant cell-based readthrough activity for several nonsense mutations derived from other nonsense-associated diseases. It is suggested that 13x has the potential to be a readthrough drug useful for the treatment of many kinds of nonsense-associated diseases.

Ubr1p-Cup9p-Ptr2p pathway involves in the sensitivity to readthrough compounds negamycin derivatives in budding yeast.

In this study, we found that dipeptide transporter Ptr2p is the putative transporter of read-through compounds (+)-negamycin derivatives TCP-126 and TCP-112, in budding yeast. Ptr2p expression and activity were correlated with the TCP-112 sensitivity, and dipeptide with high affinity to Ptr2p suppressed the TCP-112 activity. These results suggest that dipeptide transporter is one of the determinants of negamycin analogs sensitivity. Abbreviation: PTC: premature termination codon.

[A case of quadruple cancer of the liver, stomach, bladder, and ureter].

A 67-year-old man with bladder cancer who was treated with transurethral resection of bladder tumour(TUR-Bt)and chemotherapy at the age of 59 years was diagnosed as having urothelial cancer by biopsy 8 years later. Detailed examination revealed the presence of synchronous triple cancer, with hepatocellular cancer and gastric cancer. Subsequently, semi-total gastrectomy, partial hepatectomy(S6), radio frequency ablation(S5, S7), and cholecystectomy were performed. Histologically, the gastric tumor was a moderately differentiated tubular adenocarcinoma, the hepatic tumor was a moderately differentiated hepatocellular carcinoma, the bladder tumor was a transitional cell carcinoma, and the ureteral tumor was an urothelial carcinoma.

Extraanatomic bypass and coronary artery grafting for coral reef aorta.

A 63-year-old man complained of abdominal angina and intermittent claudication. Multidimensional angiography showed focal calcified obstruction of the suprarenal aorta, occlusion of the right external iliac artery, and instent restenosis in the right coronary artery. Extraanatomic bypass was performed from the ascending aorta to the left external iliac and right femoral arteries, using an 8-mm bifurcated graft, with concomitant off-pump coronary artery grafting. The patient's digestive symptoms and leg claudication disappeared.