Switchable Regioselective C-H Activation/Annulation of Acrylamides with Alkynes for the Synthesis of 2-Pyridones.

A catalyst-based switchable regioselective C-H activation/annulation of acrylamides with propargyl carbonates has been developed, delivering C5 or C6 alkenyl substituted 2-pyridones. This robust protocol proceeds with a broad substrate scope and good functional group tolerance under redox-neutral reaction conditions. More significantly, this reaction is highly effective with previously challenging unsymmetrical alkynes, including unbiased alkyl-alkyl substituted alkynes, with perfect and switchable regioselectivity. Additionally, mechanistic studies and DFT calculations were performed to shed light on the switchable regioselectivity.

Discovery of potent PROTAC degraders of Pin1 for the treatment of acute myeloid leukemia.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is overexpressed and/or overactivated in many human cancers and has been shown to play a critical role during oncogenesis. Despite the potential of Pin1 as a drug target, its successful targeting has proved to be challenging. We speculate that only blocking the enzymatic function of Pin1 with inhibitors may not be sufficient to lead to a total loss-of-function. Here, we report the discovery of P1D-34, a first-in-class and potent PROTAC degrader of Pin1, which induced Pin1 degradation with a DC50 value of 177 nM and exhibited potent degradation-dependent anti-proliferative activities in a panel of acute myeloid leukemia (AML) cell lines. In contrast, Pin1 inhibitor Sulfopin did not show activity. More significantly, P1D-34 could sensitize Bcl-2 inhibitor ABT-199 in Bcl-2 inhibitor-resistant AML cells, highlighting the potential therapeutic value of targeted Pin1 degradation for Bcl-2 inhibitor-resistant AML treatment. Further mechanism study revealed that P1D-34 led to the up-regulation of ROS pathway and down-regulation of UPR pathway to induce cell DNA damage and apoptosis. Notably, we further demonstrated that treatment with the combination formula of glucose metabolism inhibitor 2-DG and P1D-34 led to a notable synergistic anti-proliferative effect, further expanding its applicability. These data clearly reveal the practicality and importance of PROTAC as a preliminary tool compound suitable for assessment of Pin1-dependent pharmacology and a promising strategy for AML treatment.

Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3.

Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.

Immunotherapy of Epstein-Barr virus (EBV) infection and EBV-associated hematological diseases with gp350/CD89-targeted bispecific antibody.

Acute and persistent infection of Epstein-Barr virus (EBV) is associated with several life-threatening hematological disorders, including lymphoproliferative disorders (LPD), hemophagocytic lymphohistiocytosis (HLH), and chronic active Epstein-Barr virus infection (CAEBV). Currently, there are no efficacious virus-targeted therapies for EBV-driven hematological diseases. To explore the potential of phagocytosis-based immunotherapy, we created a bispecific antibody by targeting the viral envelope protein gp350 with a novel EBV-neutralizing antibody (named R1) that was paired with a monoclonal antibody against CD89 for redirecting macrophages and neutrophils. In vitro study showed that the bispecific antibody enabled efficient phagocytosis of EBV and killing of gp350 + lymphoma cells in the presence of PBMC. In vivo studies in NSG mice inoculated with EBV showed that bispecific antibody dramatically reduced the viral load in blood, solid organs and tissues. Treatment of mice implanted with EBV-harboring Raji lymphoma cells efficiently prevented tumor formation and massive metastasis to solid organs. Treatment of mice implanted with whole blood from EBV-HLH patients was effective in reducing viral levels in blood and solid organ. The gp350/CD89 bispecific antibody was highly effective in clearing EBV and immunotherapy of EBV-driven hematological diseases such as LPD and EBV-HLH.

Discovery of Highly Potent and Selective Thyroid Hormone Receptor ß Agonists for the Treatment of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive stage of nonalcoholic fatty liver disease (NAFLD) and is characterized by steatosis, inflammation, hepatocyte ballooning, and fibrosis. While there are currently no approved therapies for NASH, the thyroid hormone receptor ß (THR-ß), primarily expressed in the liver, is emerging as an effective molecular target for the treatment of NASH. However, the adverse cardiac and bone effects mediated by thyroid hormone receptor α (THR-α) need to be minimized. Herein, we reported the discovery of a series of novel THR-ß agonists featuring pyrrolo[3,2-b]pyridin-5-one skeletons based on structure-based drug design. Further optimization led to compound 15, which exhibited higher potency and selectivity for THR-ß over THR-α compared to clinical drug MGL-3196. More significantly, an excellent liver-to-serum ratio of 93:1 was observed for compound 15. We believe that the high hepatic concentration of compound 15 may result in no cardiotoxicity.

Cyclization strategy leads to highly potent Bromodomain and extra-terminal (BET) Bromodomain inhibitors for the treatment of acute liver injury.

Acute liver injury (ALI) is characteristic of abrupt hepatic dysfunction and inflammatory response, and currently the main treatment for ALI is merely supportive rather than curative. Therefore, the development of novel and effective therapeutic strategies for ALI therapy is highly desirable. The emerging biological understanding of the role of BET Bromodomains has opened up an exciting opportunity to develop potent BET Bromodomain inhibitors as an effective therapeutic strategy for the treatment of acute liver injury. Herein, we synthesized a series of potent BET Bromodomain inhibitors with a tetracyclic scaffold, exemplified by compound 28 which showed good in vitro anti-inflammatory activity and good therapeutic effects in the LPS-induced acute liver injury model without obvious cytotoxicity, suggesting that compound 28 is a highly promising candidate worthy for further development.

Development of Small-Molecule Fluorescent Probes Targeting Enzymes.

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.

Discovery of a Potent and Selective Degrader for USP7.

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.

Discovery of novel Thieno[2,3-d]imidazole derivatives as agonists of human STING for antitumor immunotherapy using systemic administration.

The activation of stimulator of interferon genes (STING) signaling pathways plays an important role in the innate immune response. Although several STING agonists have been developed recently, the majority of clinical CDN STING agonists are administered by intratumoral (IT) injection. Therefore, there remains a need to develop diverse non-CDN small-molecule STING agonists with systemic administration. Herein, by using a scaffold hopping strategy, we designed a series of thieno [2,3-d]imidazole derivatives as novel STING agonists. Further structure-activity relationship study and optimization led to the discovery of compound 45 as a highly potent human STING agonist with an EC50 value of 1.2 nM. Compound 45 was found to bind to multiple human STING isoforms and accordingly activated the downstream TBK1/IRF3 and NF-κB signaling pathways in the reporter cells bearing with different STING isoforms. The activation on STING signaling pathway was abolished in the STING knock-out cells, indicating that it is a specific STING agonist. Compound 45 significantly inhibited the tumor growth in allograft 4T1 and CT26 tumor models by systemic administration, and more significantly, 45 was able to induce tumor regression in CT26 tumor model without inducing weight loss, suggesting that compound 45 is a highly promising candidate worthy for further development.

Rhodium(III)-Catalyzed Asymmetric 1,2-Carboamidation of Alkenes Enables Access to Chiral 2,3-Dihydro-3-benzofuranmethanamides.

Through the initial screening and further rational design of chiral cyclopentadienyl ligands, a chiral rhodium-catalyzed enantioselective 1,2-carboamidation of aromatic tethered alkenes was developed, enabling the asymmetric preparation of various chiral 2,3-dihydro-3-benzofuranmethanamides with an enantioenriched all-carbon quaternary center at the ß position of amide. This robust transformation has a broad functional group tolerance, excellent enantioselectivities (up to 98.5:1.5 er), and a mild reaction conditions, releasing CO2 as the single byproduct.

Highly Potent Immunotoxins Targeting the Membrane-distal N-lobe of GPC3 for Immunotherapy of Hepatocellular Carcinoma.

Glypican-3 (GPC3) has become a compelling target for immunotherapy of hepatocellular carcinoma, including antibody-drug conjugate (ADC), and ADC-like immunotoxin. To investigate the impact of epitopes on the potency of ADCs, current study generated a large panel of chicken monoclonal antibodies (mAbs) that targeted 12 different and over-lapping epitopes on GPC3. These mAbs demonstrated a very high affinity with Kd values in the range of 10-9-10-14 M, and the highest affinity (Kd value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (Kd value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with Tm values in the range of 45-82 °C. As a proof-of-concept study for ADC, we made immunotoxins (scFv fused with PE24, the 24-kDa cytotoxic domain of Pseudomonas exotoxin A) based on these mAbs, and we found that immunotoxins targeting the N-lobe of GPC3 were overall much more potent than those targeting the C-lobe and other locations. One representative N-lobe-targeting immunotoxin J80A-PE24 demonstrated 3 to 13-fold more potency than the hitherto best immunotoxin HN3-PE24 that was previously developed. J80A-PE24 could suppress tumor growth much greater than HN3-PE24 in a xenograft mouse model. Combination of J80A-PE24 with an angiogenesis inhibitor FGF401 showed additive effect, which dramatically shrank tumor growth. Our work demonstrated that, due to high affinity, excellent thermostability and potency, chicken mAbs targeting the N-lobe of GPC3 are appealing candidates to develop potent ADCs for immunotherapy of liver cancer.

P300/CBP inhibition sensitizes mantle cell lymphoma to PI3Kδ inhibitor idelalisib.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder lacking reliable therapies. PI3K pathway contributes to the pathogenesis of MCL, serving as a potential target. However, idelalisib, an FDA-approved drug targeting PI3Kδ, has shown intrinsic resistance in MCL treatment. Here we report that a p300/CBP inhibitor, A-485, could overcome resistance to idelalisib in MCL cells in vitro and in vivo. A-485 was discovered in a combinational drug screening from an epigenetic compound library containing 45 small molecule modulators. We found that A-485, the highly selective catalytic inhibitor of p300 and CBP, was the most potent compound that enhanced the sensitivity of MCL cell line Z-138 to idelalisib. Combination of A-485 and idelalisib remarkably decreased the viability of three MCL cell lines tested. Co-treatment with A-485 and idelalisib in Maver-1 and Z-138 MCL cell xenograft mice for 3 weeks dramatically suppressed the tumor growth by reversing the unsustained inhibition in PI3K downstream signaling. We further demonstrated that p300/CBP inhibition decreased histone acetylation at RTKs gene promoters and reduced transcriptional upregulation of RTKs, thereby inhibiting the downstream persistent activation of MAPK/ERK signaling, which also contributed to the pathogenesis of MCL. Therefore, additional inhibition of p300/CBP blocked MAPK/ERK signaling, which rendered maintaining activation to PI3K-mTOR downstream signals p-S6 and p-4E-BP1, thus leading to suppression of cell growth and tumor progression and eliminating the intrinsic resistance to idelalisib ultimately. Our results provide a promising combination therapy for MCL and highlight the potential use of epigenetic inhibitors targeting p300/CBP to reverse drug resistance in tumor.

Design, synthesis and biological evaluation of a novel spiro oxazolidinedione as potent p300/CBP HAT inhibitor for the treatment of ovarian cancer.

Histone acetylation is one of the most essential parts of epigenetic modification, mediating a variety of complex biological functions. In these procedure, p300/CBP could catalyze the acetylation of lysine 27 on histone 3 (H3K27ac), and had been reported to mediate tumorigenesis and development in a variety of tumors by enhancing chromatin transcription activity. Ovarian cancer, as an extremely malignant tumor, has also been observed to undergo abnormal acetylation of histones. However, whether the treatment of ovarian cancer could be achieved by inhibiting the acetylation activity of p300/CBP on H3K27 has not been well investigated. In this article, we modified the structure of p300/CBP HAT domain inhibitor A-485 and obtained a highly active small molecule known as 13f, which has an IC50 value of 0.49 nM for inhibiting the in vitro enzyme activity of p300, as well as the anti-proliferation IC50 value on ovarian cancer cell line OVCAR-3 was 153 nM. In addition, 13f had strong acetylase family selectivity, good metabolic stability and promising in vivo anti-tumor activity in OVCAR-3 xenograft model. The discovery of 13f revealed a more active chemical entity of the HATs domain of p300/CBP and provided a novel idea for the application of epigenetic inhibitors in the treatment of ovarian cancer.

Optical Mapping of Brain Activity Underlying Directionality and Its Modulation by Expertise in Mandarin/English Interpreting.

Recent neuroimaging research has suggested that unequal cognitive efforts exist between interpreting from language 1 (L1) to language 2 (L2) compared with interpreting from L2 to L1. However, the neural substrates that underlie this directionality effect are not yet well understood. Whether directionality is modulated by interpreting expertise also remains unknown. In this study, we recruited two groups of Mandarin (L1)/English (L2) bilingual speakers with varying levels of interpreting expertise and asked them to perform interpreting and reading tasks. Functional near-infrared spectroscopy (fNIRS) was used to collect cortical brain data for participants during each task, using 68 channels that covered the prefrontal cortex and the bilateral perisylvian regions. The interpreting-related neuroimaging data was normalized by using both L1 and L2 reading tasks, to control the function of reading and vocalization respectively. Our findings revealed the directionality effect in both groups, with forward interpreting (from L1 to L2) produced more pronounced brain activity, when normalized for reading. We also found that directionality was modulated by interpreting expertise in both normalizations. For the group with relatively high expertise, the activated brain regions included the right Broca's area and the left premotor and supplementary motor cortex; whereas for the group with relatively low expertise, the activated brain areas covered the superior temporal gyrus, the dorsolateral prefrontal cortex (DLPFC), the Broca's area, and visual area 3 in the right hemisphere. These findings indicated that interpreting expertise modulated brain activation, possibly because of more developed cognitive skills associated with executive functions in experienced interpreters.

Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders.

SHP2, a non-receptor tyrosine phosphatase, plays a pivotal role in numerous oncogenic cell-signaling cascades like RAS-ERK, PI3K-AKT and JAK-STAT. On the other hand, proteolysis targeting chimera (PROTAC) has emerged as a promising strategy for the degradation of disease-related protein of interest (POI). SHP2 degradation via the PROTAC strategy will provide an alternative startegy for SHP2-mediated cancer therapy. Herein we described the design, synthesis and evaluation of a series of thalidomide-based heterobifunctional molecules and identified 11(ZB-S-29) as the highly efficient SHP2 degrader with a DC50 of 6.02 nM. Further mechanism investigation illustrated that 11 came into function through targeted SHP2 protein degradation.

Rhodium-Catalyzed Twofold Unsymmetrical C-H Alkenylation-Annulation/Thiolation Reaction To Access Thiobenzofurans.

A Rh(III)-catalyzed twofold unsymmetrical C-H alkenylation-annulation/thiolation reaction has been developed, enabling the straightforward and efficient synthesis of various thiobenzofurans in one step. This robust protocol proceeds with a broad substrate scope and good functional group tolerance under relatively mild reaction conditions.

Targeting p300/CBP Attenuates Hepatocellular Carcinoma Progression through Epigenetic Regulation of Metabolism.

Targeting epigenetics in cancer has emerged as a promising anticancer strategy. p300/CBP is a central regulator of epigenetics and plays an important role in hepatocellular carcinoma (HCC) progression. Tumor-associated metabolic alterations contribute to the establishment and maintenance of the tumorigenic state. In this study, we used a novel p300 inhibitor, B029-2, to investigate the effect of targeting p300/CBP in HCC and tumor metabolism. p300/CBP-mediated acetylation of H3K18 and H3K27 increased in HCC tissues compared with surrounding noncancerous tissues. Conversely, treatment with B029-2 specifically decreased H3K18Ac and H3K27Ac and displayed significant antitumor effects in HCC cells in vitro and in vivo. Importantly, ATAC-seq and RNA-seq integrated analysis revealed that B029-2 disturbed metabolic reprogramming in HCC cells. Moreover, B029-2 decreased glycolytic function and nucleotide synthesis in Huh7 cells by reducing H3K18Ac and H3K27Ac levels at the promoter regions of amino acid metabolism and nucleotide synthesis enzyme genes, including PSPH, PSAT1, ALDH18A1, TALDO1, ATIC, and DTYMK. Overexpression of PSPH and DTYMK partially reversed the inhibitory effect of B029-2 on HCC cells. These findings suggested that p300/CBP epigenetically regulates the expression of glycolysis-related metabolic enzymes through modulation of histone acetylation in HCC and highlights the value of targeting the histone acetyltransferase activity of p300/CBP for HCC therapy. SIGNIFICANCE: This study demonstrates p300/CBP as a critical epigenetic regulator of glycolysis-related metabolic enzymes in HCC and identifies the p300/CBP inhibitor B029-2 as a potential therapeutic strategy in this disease.

Experience-Dependent Counselor-Client Brain Synchronization during Psychological Counseling.

The role of the counselor's experience in building an alliance with the clients remains controversial. Recently, the expanding nascent studies on interpersonal brain synchronization (IBS) using functional near-infrared spectroscopy (fNIRS) on human subjects have hinted at the possible neural substrates underlying the relationship qualities between the counselor-client dyads. Our study assessed the clients' self-report working alliance (WA) as well as simultaneously measured IBS by fNIRS in 14 experienced versus 16 novice counselor-client dyads during the first integrative-orientation psychological counseling session. We observed that synchronous brain activity patterns were elicited from the right temporo-parietal junction (rTPJ) across counselor-client dyads. Furthermore, such IBS, together with alliance quality, was especially evident when counselors had more psychotherapy experience. Time-lagged counselor-client brain synchronization might co-vary with the alliance (goal component) when the client's brain activity preceded that of the counselor. These findings favor the notion that the IBS between counselor-client associated with the WA is an experience-dependent phenomenon, suggesting that a potential adaptive mechanism is embedded in psychological counseling.

Complete genome sequence of an isolate of duck enteritis virus from China.

Here, we present the complete genomic sequence of duck enteritis virus (DEV) strain SD, isolated in China in 2012. The virus was virulent in experimentally infected 2-month-old ducks. The DEV SD genome is 160,945 base pairs (bp) in length. The viral genome sequence, when compared to that of strain DEV CSC, which was isolated in 1962, showed three discontinuous deletions of 101 bp, 48 bp and 417 bp within the inverted repeats. A comparison of the amino acid (aa) sequences of all ORFs of the CSC and SD isolates demonstrated an11-aa deletion, two single-aa deletions, and one single-aa deletion in LORF3, UL47, UL4, respectively. Moreover, 38 single aa variations were also detected in 24 different ORFs. These results will further advance our understanding of the genetic variations involved in evolution.

Discovery of 8-Methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one Derivatives as Highly Potent and Selective Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitors.

The bromodomain and extra-terminal (BET) family proteins have recently emerged as promising drug targets for cancer therapy. In this study, identification of an 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one fragment (47) as a new binder to the BET bromodomains and the subsequent incorporation of fragment 47 to the scaffold of ABBV-075, which recently entered Phase I clinical trials, enabled the generation of a series of highly potent BET bromodomain inhibitors. Further druggability optimization led to the discovery of compound 38 as a potential preclinical candidate. Significantly, compared with ABBV-075, which exhibits a 63-fold selectivity for BRD4(1) over EP300, compound 38 demonstrates an excellent selectivity for the BET bromodomain family over other bromodomains, with an ∼1500-fold selectivity for BRD4(1) over EP300. Orally administered 38 achieves a complete inhibition of tumor growth with a tumor growth inhibition (TGI) of 99.7% accompanied by good tolerability.