The COVID-19 pandemic caused by the SARS-CoV-2 virus continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either Boceprevir or Telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro with IC50 values ranging from 7.6 to 748.5 nM. The co-crystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a SARS-CoV-2 infection transgenic mouse model, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

Human Ph + leukemias are caused by the BCR-ABL1 oncogene and include CML and B-ALL. CML has a triphasic clinical course, with chronic and accelerated phases followed by a terminal blast crisis phase resembling acute leukemia, in which myeloid or lymphoid blasts fail to differentiate. Together, lymphoid blast crisis of CML and Ph + B-ALL account for 20% of adult cases and 5% of pediatric cases of acute lymphoblastic leukemia. One-half of adult and 20% of pediatric BALLs express the p210 isoform of Bcr-Abl, and the remainder express the p190 isoform 1,2 . Whereas the Abl tyrosine kinase inhibitor imatinib mesylate induces a complete hematologic response in nearly all individuals with chronic phase CML 3 , imatinib is much less effective in treating CML blast crisis and Ph + B-ALL 2 owing to acquired resistance [4][5][6][7] . Drugs that target essential signaling molecules downstream of Bcr-Abl may help overcome or prevent imatinib resistance.Bcr-Abl activates multiple signaling pathways, including Ras, MAPK, STAT, JNK/SAPK, PI-3 kinase, NF-kB and c-Myc 8 . Furthermore, in myeloid cell lines Bcr-Abl activates the Src family kinases Lyn and Hck 9 . Multiple domains of Bcr-Abl interact with and activate Src kinases independently of Bcr-Abl kinase activity [10][11][12] , and studies with dominant-negative mutants and Src inhibitors suggest that Src kinases may contribute to the proliferation and survival of myeloid cell lines expressing Bcr-Abl in vitro [13][14][15] . Results in cell lines expressing Bcr-Abl often do not correlate with leukemogenesis in vivo 16 , emphasizing the need to evaluate signaling pathways in an animal model of leukemia. We developed accurate and quantitative mouse models of chronic phase CML 17 and Ph + B-ALL 17,18 . Here, we use these models to show that Src kinases are required for the induction of B-ALL by Bcr-Abl but are dispensable for induction of CMLlike myeloproliferative disease. RESULTS Bcr-Abl activates Lyn, Hck and Fgr in pre-B cellsBcr-Abl interacts with and activates the Src kinases Lyn and Hck in myeloid cells 9 . We investigated whether Bcr-Abl also activates Src kinases in pre-B lymphoid cells. We found Bcr-Abl protein and abundant tyrosine-phosphorylated cell proteins in BL-2 cells (isolated from a mouse with Bcr-Abl-induced B-ALL) and in ENU48515 cells (an N-ethyl-N-nitrosourea (ENU)-induced pre-B leukemia mouse line) transduced with BCR-ABL1 retrovirus, but not in vector-transduced cells (Fig. 1a). Of the eight Src family kinases expressed in hematopoetic cells (Blk, Fgr, Fyn, Hck, Lck, Lyn, c-Src and Yes), parental ENU48515 cells had moderate levels of constitutively activate Fyn and Lyn, and Bcr-Abl expression increased the activation of Lyn and also activated five other Src kinases (Blk, Fgr, Hck, Lck and c-Src), as indicated by increased tyrosine phosphorylation (Fig. 1b). Phosphorylation of Fyn was not increased with BcrAbl expression in ENU48515 cells, and Yes was not expressed. In primary leukemic cells isolated from peripheral blood of mice with

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositolrequiring enzyme 1␣ (IRE1␣) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1␣- IntroductionTreatment for multiple myeloma (MM) has remarkably improved because of novel agents, such as bortezomib, thalidomide, and lenalidomide. [1][2][3] However, MM remains incurable, and nextgeneration novel agents are urgently needed. Because of high levels of endoplasmic reticulum (ER) stress and adaptation by the unfolded protein response (UPR), targeting signaling by the UPR and blocking this key survival pathway represent a new therapeutic strategy. In mammalian cells, protein folding is proportionally fine-tuned to the metabolic state of the cell within its microenvironment. Extracellular insults, such as low nutrients, hypoxia, and multiple drugs, result in the accumulation of misfolded proteins in the ER, thereby causing ER stress and initiating the UPR. 4 The UPR in turn increases the biosynthetic capacity and decreases the biosynthetic burden of the ER, to maintain cellular homeostasis. However, when the stress cannot be compensated by the UPR, cellular apoptosis occurs. 5 The UPR consists of 3 branches of signaling pathways, which initiate from 3 ER transmembrane proteins: inositol-requiring enzyme 1␣ (IRE1␣), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). In the resting state, these proteins are associated with molecular chaperone BiP/GRP78 in the ER. However, when unfolded proteins accumulate in the ER, BiP/GRP78 dissociates from them, thereby inducing UPR signaling. 6 In the UPR, IRE1␣ is activated by oligomerization and autophosphorylation, resulting in activation of its endoribonuclease to cleave and initiate splicing of the X-box binding protein 1 (XBP1) mRNA. A 26-nucleotide intron from XBP1 is removed by activated IRE1␣ endoribonuclease, resulting in a translational frame-shift to modify unspliced XBP1 (XBP1u: inactive) into spliced XBP1 (XBP1s: active). 7 XBP1 is a unique transcription factor that regulates genes responsible for ER-associated degradation (ERAD), such as EDEM, and those responsible for promoting protein folding, such as p58IPK and other ER chaperones. 8 Thus, IRE1␣-XBP1 pathway has a prosurvival role in the UPR. However, under conditions of prolonged and uncompensated stress, the UPR leads to cellular apoptosis, known as the terminal UPR. The proapoptotic transcription factor CHOP, also known as GADD153, is induced via PERK and ATF6 pathways. CHOP causes downregulation of BCL2, thereby leading to caspase-dependent apoptosis. 9 IRE1␣ also has a proapoptotic role: it binds TRAF2 and activates ASK1, which causes JNK activation, thereby leading to caspase-dependent apoptosis. 10 ...

It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph ؉ ) chronic myeloid leukemia (CML) in chronic phase but not Ph ؉ B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABLactivated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph ؉ leukemia.dasatinib ͉ imatinib ͉ SRC kinases