Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Research progress on the role of orphan receptor GPR139 in neuropsychiatric behaviours.

The study of orphan G protein-coupled receptors (GPCRs) holds much promise for increasing our understanding of neuropsychiatric diseases and for the development of new therapeutic strategies for these diseases. GPR139 is an orphan GPCR expressed in the central nervous system, especially in areas of the brain that control movement, motivation, and reward, and those that regulate neuropsychiatric behaviour. This review provides information about the discovery, tissue expression, signal transduction pathways, and physiological functions of GPR139, as well as how GPR139 interacts with other GPCRs, which form heteromeric complexes that affect their pharmacology and function. We also discuss the utility and therapeutic potential of ligands that target GPR139, including the pharmacological properties of reported agonists and antagonists. Finally, we highlight the pathologic role of GPR139 in neuropsychiatric behaviour and its potential as a therapeutic target in neuropsychiatric disorders.

Heterodimerization of apelin and opioid receptor-like 1 receptors mediates apelin-13-induced G protein biased signaling.

The apelin receptor (APJ) and the opioid-related nociceptin receptor 1 (ORL1) are family A G protein-coupled receptors that participate in a variety of physiological processes. The distribution and function of APJ and ORL1 in the nervous system and peripheral tissues are similar; however, the detailed mechanism of how these two receptors modulate signaling and physiological effects remains unclear. Here, we examined whether APJ and ORL1 form dimers, and investigated signal transduction pathways. The endogenous co-expression of APJ and ORL1 in SH-SY5Y cells was confirmed by western blotting and RT-PCR. Bioluminescence and fluorescence resonance energy transfer assays, as well as a proximity ligation assay and co-immunoprecipitation experiments, demonstrated that APJ and ORL1 heterodimerize in HEK293 cells. We found that the APJ-ORL1 heterodimer is selectively activated by apelin-13, which causes the dimer to couple to Gαi proteins and reduce the recruitment of GRKs and ß-arrestins to the dimer. We showed that the APJ-ORL1 dimer exhibits biased signaling, in which G protein-dependent signaling pathways override ß-arrestin-dependent signaling pathways. Our results demonstrate that the structural interface of the APJ-ORL1 dimer switches from transmembrane domain TM1/TM2 in the inactive state to TM5 in the active state. We used mutational analysis and BRET assays to identify key residues in TM5 (APJ L2185.55, APJ I2245.61, and ORL1 L2295.52) responsible for the receptor-receptor interaction. These results provide important information on the APJ-ORL1 heterodimer and may assist the design of new drugs targeting biased signaling pathways for treatment of pain and cardiovascular and metabolic diseases.

Apelin-13 prevents the effects of oxygen-glucose deprivation/reperfusion on bEnd.3 cells by inhibiting AKT-mTOR signaling.

Autophagy plays works by degrading misfolded proteins and dysfunctional organelles and maintains intracellular homeostasis. Apelin-13 has been investigated as an agent that might protect the blood-brain barrier (BBB) from cerebral ischemia/reperfusion (I/R) injury. In this study, we examined whether apelin-13 protects cerebral microvascular endothelial cells, important components of the BBB, from I/R injury by regulating autophagy. To mimic I/R injury, the mouse cerebral microvascular endothelia l cell line bEnd 3 undergoes the process of oxygen and glucose deprivation and re feeding in the process of culture. Cell viability was detected using a commercial kit, and cell migration was monitored by in vitro scratch assay. The tight junction (TJ) proteins ZO-1 and occludin; the autophagy markers LC3 II, beclin 1, and p62; and components of the AKT-mTOR signaling pathway were detected by Western blotting and immunofluorescence. To confirm the role of autophagy in OGD/R and the protective effect of apelin-13, we treated the cells with 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy. Our results demonstrated that OGD/R increased autophagic activity but decreased viability, abundance of TJs, and migration. Viability and TJ abundance were further reduced when the OGD/R group was treated with 3-MA. These results indicated that bEnd.3 upregulates autophagy to ameliorate the effects of OGD/R injury on viability and TJs, but that the autophagy induced by OGD/R alone is not sufficient to protect against the effect on cell migration. Treatment of OGD/R samples with apelin-13 markedly increased viability, TJ abundance, and migration, as well as autophagic activity, whereas 3-MA inhibited this increase, suggesting that apelin-13 exerted its protective effects by upregulating autophagy.

The transmembrane domains of GPCR dimers as targets for drug development.

G-protein-coupled receptors (GPCRs) can form homodimers or heterodimers that modulate specific signal transduction pathways to regulate a wide range of physiological and pathological functions. As such, GPCR dimers are novel drug targets for disorders including depression, hypertension, diabetes, and vascular dementia. The interaction between two receptors in a GPCR dimer involves a conformational change in the transmembrane domain (TMD). It has been demonstrated that the TMD has an important role in GPCR dimer formation and stability in vitro and in vivo. Moreover, increasing evidence shows that the TMD of GPCRs affects the function of dimers. Therefore, the TMD of GPCRs is an emerging target for the development of drugs to treat diseases that involve GPCR dimerization.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Do COVID-19 CT features vary between patients from within and outside mainland China? Findings from a meta-analysis.

Background: Chest computerized tomography (CT) plays an important role in detecting patients with suspected coronavirus disease 2019 (COVID-19), however, there are no systematic summaries on whether the chest CT findings of patients within mainland China are applicable to those found in patients outside. Methods: Relevant studies were retrieved comprehensively by searching PubMed, Embase, and Cochrane Library databases before 15 April 2022. Quality assessment of diagnostic accuracy studies (QUADAS) was used to evaluate the quality of the included studies, which were divided into two groups according to whether they were in mainland China or outside. Data on diagnostic performance, unilateral or bilateral lung involvement, and typical chest CT imaging appearances were extracted, and then, meta-analyses were performed with R software to compare the CT features of COVID-19 pneumonia between patients from within and outside mainland China. Results: Of the 8,258 studies screened, 19 studies with 3,400 patients in mainland China and 14 studies with 554 outside mainland China were included. Overall, the risk of quality assessment and publication bias was low. The diagnostic value of chest CT is similar between patients from within and outside mainland China (93, 91%). The pooled incidence of unilateral lung involvement (15, 7%), the crazy-paving sign (31, 21%), mixed ground-glass opacities (GGO) and consolidations (51, 35%), air bronchogram (44, 25%), vascular engorgement (59, 33%), bronchial wall thickening (19, 12%), and septal thickening (39, 26%) in patients from mainland China were significantly higher than those from outside; however, the incidence rates of bilateral lung involvement (75, 84%), GGO (78, 87%), consolidations (45, 58%), nodules (12, 17%), and pleural effusion (9, 15%) were significantly lower. Conclusion: Considering that the chest CT features of patients in mainland China may not reflect those of the patients abroad, radiologists and clinicians should be familiar with various CT presentations suggestive of COVID-19 in different regions.

Mechanistic insights on novel small molecule allosteric activators of cGMP-dependent protein kinase PKG1α.

cGMP-dependent protein kinase (PKG) represents a compelling drug target for treatment of cardiovascular diseases. PKG1 is the major effector of beneficial cGMP signaling which is involved in smooth muscle relaxation and vascular tone, inhibition of platelet aggregation and signaling that leads to cardioprotection. In this study, a novel piperidine series of activators previously identified from an ultrahigh-throughput screen were validated to directly bind partially activated PKG1α and subsequently enhance its kinase activity in a concentration-dependent manner. Compounds from initial optimization efforts showed an ability to activate PKG1α independent of the endogenous activator, cGMP. We demonstrate these small molecule activators mimic the effect of cGMP on the kinetic parameters of PKG1α by positively modulating the KM of the peptide substrate and negatively modulating the apparent KM for ATP with increase in catalytic efficiency, kcat. In addition, these compounds also allosterically modulate the binding affinity of cGMP for PKG1α by increasing the affinity of cGMP for the high-affinity binding site (CNB-A) and decreasing the affinity of cGMP for the low-affinity binding site (CNB-B). We show the mode of action of these activators involves binding to an allosteric site within the regulatory domain, near the CNB-B binding site. To the best of our knowledge, these are the first reported non-cGMP mimetic small molecules shown to directly activate PKG1α. Insights into the mechanism of action of these compounds will enable future development of cardioprotective compounds that function through novel modes of action for the treatment of cardiovascular diseases.

Real-Word Effectiveness of Global COVID-19 Vaccines Against SARS-CoV-2 Variants: A Systematic Review and Meta-Analysis.

Background: Currently, promoted vaccinations against SARS-CoV-2 are being given out globally. However, the occurrence of numerous COVID-19 variants has hindered the goal of rapid mitigation of the COVID-19 pandemic by effective mass vaccinations. The real-word effectiveness of the current vaccines against COVID-19 variants has not been assessed by published reviews. Therefore, our study evaluated the overall effectiveness of current vaccines and the differences between the various vaccines and variants. Methods: PubMed, Embase, Cochrane Library, medRxiv, bioRxiv, and arXiv were searched to screen the eligible studies. The Newcastle-Ottawa scale and the Egger test were applied to estimate the quality of the literature and any publication bias, respectively. The pooled incident rates of different variants after vaccination were estimated by single-arm analysis. Meanwhile, the pooled efficacies of various vaccines against variants were evaluated by two-arm analysis using odds ratios (ORs) and vaccine effectiveness (VE). Results: A total of 6,118 studies were identified initially and 44 articles were included. We found that the overall incidence of variants post first/second vaccine were 0.07 and 0.03, respectively. The VE of the incidence of variants post first vaccine between the vaccine and the placebo or unvaccinated population was 40% and post second vaccine was 96%, respectively. The sub-single-arm analysis showed a low prevalence rate of COVID-19 variants after specific vaccination with the pooled incidence below 0.10 in most subgroups. Meanwhile, the sub-two-arm analysis indicated that most current vaccines had a good or moderate preventive effect on certain variants considering that the VE in these subgroups was between 66 and 95%, which was broadly in line with the results of the sub-single-arm analysis. Conclusion: Our meta-analysis shows that the current vaccines that are used globally could prevent COVID-19 infection and restrict the spread of variants to a great extent. We would also support maximizing vaccine uptake with two doses, as the effectiveness of which was more marked compared with one dose. Although the mRNA vaccine was the most effective against variants according to our study, specific vaccines should be taken into account based on the local dominant prevalence of variants.

Micronutrient Level Is Negatively Correlated with the Neutrophil-Lymphocyte Ratio in Patients with Severe COVID-19.

Aim: To explore the potential relationship between NLR and micronutrient deficiency in patients with severe COVID-19 infection. Methods: Sixteen patients were categorized into the mild group (mild COVID-19) and severe group (severe COVID-19) based on the guideline of the management of COVID-19. The lactate dehydrogenase (LDH); superoxide dismutase (SOD), the inflammatory markers (neutrophil lymphocyte ratio (NLR)), erythrocyte sedimentation rate (ESR), c-reactive protein (CRP), selenium (Se), iron (Fe), zinc (Zn), nickel (Ni), copper (Cu), chromium (Cr), cadmium (Cd), arsenic (As), and manganese (Mn) were measured in the blood. Results: Compared to the mild group, the NLR (P < 0.05) and the level of Se (P < 0.01), Fe (P < 0.05), and Zn (P < 0.05) were significantly decreased in the severe group. The level of Se, Fe, and Zn was significantly correlated to NLR levels. Furthermore, close positive correlation was found between NLR and severity of COVID-19. Conclusion: The micronutrient deficiency in the blood is associated with NLR in the severity of COVID-19 patients.

SHR1032, a novel STING agonist, stimulates anti-tumor immunity and directly induces AML apoptosis.

Stimulator of interferon genes (STING) activation induces type I interferons and pro-inflammatory cytokines which stimulate tumor antigen cross presentation and the adaptive immune responses against tumor. The first-generation of STING agonists, cyclic di-nucleotide (CDN), mimicked the endogenous STING ligand cyclic guanosine monophosphate adenosine monophosphate, and displayed limited clinical efficacy. Here we report the discovery of SHR1032, a novel small molecule non-CDN STING agonist. Compared to the clinical CDN STING agonist ADU-S100, SHR1032 has much higher activity in human cells with different STING haplotypes and robustly induces interferon ß (IFNß) production. When dosed intratumorally, SHR1032 induced strong anti-tumor effects in the MC38 murine syngeneic tumor model. Pharmacodynamic studies showed induction of IFNß, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) in the tumors and, to a lower extent, in the plasma. More importantly, we found SHR1032 directly causes cell death in acute myeloid leukemia (AML) cells. In conclusion, our findings demonstrate that in addition to their established ability to boost anti-tumor immune responses, STING agonists can directly eradicate AML cells, and SHR1032 may present a new and promising therapeutic agent for cancer patients.

Discovery of novel spiro compound as RAF kinase inhibitor with in vitro potency against KRAS mutant cancer.

The development of RAF inhibitors targeting cancers with wild type RAF kinase and/or RAS mutation has been challenging due to the paradoxical activation of the RAS-RAF-MEK-ERK cascade following RAF inhibitor treatment. Herein is the discovery and optimization of a series of RAF inhibitors with a novel spiro structure. The most potent spiro molecule 9 showed excellent in vitro potency against b/c RAF enzymes and RAS mutant H358 cancer cells with minimal paradoxical RAF signaling activation. Compound 9 also exhibited good drug-like properties as demonstrated by in vitro cytochrome P450 (CYP), liver microsome stability (LMS) data and moderate oral pharmacokinetics (PK) profiles in rat and mouse.

Lead Optimization to Advance Protease-Activated Receptor-1 Antagonists in Early Discovery.

Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.

Disruption of 5-hydroxytryptamine 1A receptor and orexin receptor 1 heterodimer formation affects novel G protein-dependent signaling pathways and has antidepressant effects in vivo.

G protein-coupled receptor (GPCR) heterodimers are new targets for the treatment of depression. Increasing evidence supports the importance of serotonergic and orexin-producing neurons in numerous physiological processes, possibly via a crucial interaction between 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 1 (OX1R). However, little is known about the function of 5-HT1AR/OX1R heterodimers. It is unclear how the transmembrane domains (TMs) of the dimer affect its function and whether its modulation mediates antidepressant-like effects. Here, we examined the mechanism of 5-HT1AR/OX1R dimerization and downstream G protein-dependent signaling. We found that 5-HT1AR and OX1R form constitutive heterodimers that induce novel G protein-dependent signaling, and that this heterodimerization does not affect recruitment of ß-arrestins to the complex. In addition, we found that the structural interface of the active 5-HT1AR/OX1R dimer transforms from TM4/TM5 in the basal state to TM6 in the active conformation. We also used mutation analyses to identify key residues at the interface (5-HT1AR R1514.40, 5-HT1AR Y1985.41, and OX1R L2305.54). Injection of chronic unpredictable mild stress (CUMS) rats with TM4/TM5 peptides improved their depression-like emotional status and decreased the number of endogenous 5-HT1AR/OX1R heterodimers in the rat brain. These antidepressant effects may be mediated by upregulation of BDNF levels and enhanced phosphorylation and activation of CREB in the hippocampus and medial prefrontal cortex. This study provides evidence that 5-HT1AR/OX1R heterodimers are involved in the pathological process of depression. Peptides including TMs of the 5-HT1AR/OX1R heterodimer interface are candidates for the development of compounds with fast-acting antidepressant-like effects.

Actively implementing an evidence-based feeding guideline for critically ill patients (NEED): a multicenter, cluster-randomized, controlled trial.

BACKGROUND: Previous cluster-randomized controlled trials evaluating the impact of implementing evidence-based guidelines for nutrition therapy in critical illness do not consistently demonstrate patient benefits. A large-scale, sufficiently powered study is therefore warranted to ascertain the effects of guideline implementation on patient-centered outcomes. METHODS: We conducted a multicenter, cluster-randomized, parallel-controlled trial in intensive care units (ICUs) across China. We developed an evidence-based feeding guideline. ICUs randomly allocated to the guideline group formed a local "intervention team", which actively implemented the guideline using standardized educational materials, a graphical feeding protocol, and live online education outreach meetings conducted by members of the study management committee. ICUs assigned to the control group remained unaware of the guideline content. All ICUs enrolled patients who were expected to stay in the ICU longer than seven days. The primary outcome was all-cause mortality within 28 days of enrollment. RESULTS: Forty-eight ICUs were randomized to the guideline group and 49 to the control group. From March 2018 to July 2019, the guideline ICUs enrolled 1399 patients, and the control ICUs enrolled 1373 patients. Implementation of the guideline resulted in significantly earlier EN initiation (1.20 vs. 1.55 mean days to initiation of EN; difference - 0.40 [95% CI - 0.71 to - 0.09]; P = 0.01) and delayed PN initiation (1.29 vs. 0.80 mean days to start of PN; difference 1.06 [95% CI 0.44 to 1.67]; P = 0.001). There was no significant difference in 28-day mortality (14.2% vs. 15.2%; difference - 1.6% [95% CI - 4.3% to 1.2%]; P = 0.42) between groups. CONCLUSIONS: In this large-scale, multicenter trial, active implementation of an evidence-based feeding guideline reduced the time to commencement of EN and overall PN use but did not translate to a reduction in mortality from critical illness. TRIAL REGISTRATION: ISRCTN, ISRCTN12233792 . Registered November 20th, 2017.

Discovery of spiro amide SHR902275: A potent, selective, and efficacious RAF inhibitor targeting RAS mutant cancers.

The RAS-RAF-MEK-ERK signaling pathway plays a key role to regulate multiple cellular functions. Acquired resistance to the first-generation RAF inhibitors that only targeted the bRAFV600E mutation prompted the need for a new generation of RAF inhibitors to target cancers bearing mutant RAS and wild type RAF activity by inhibition of paradoxical activation. Starting from the company's previously reported RAF inhibitor 1, extensive drug potency and drug-like properties optimizations led to the discovery of molecule 33 (SHR902275) with greatly improved in vitro potency and solubility. Molecule 33 exhibited good DMPK (Drug Metabolism and Pharmacokinetics) properties, excellent permeability, and outstanding mouse/rat oral PK. It was further evaluated in an in vivo RAS mutant Calu6 xenograft mouse model and demonstrated dose dependent efficacy. To achieve high exposure in a toxicity study, pro-drug 48 was also explored.

Discovery of 2-(Ortho-Substituted Benzyl)-Indole Derivatives as Potent and Orally Bioavailable RORγ Agonists with Antitumor Activity.

RORγ is a dual-functional drug target, which involves not only induction of inflammation but also promotion of cancer immunity. The development of agonists of RORγ promoting Th17 cell differentiation could provide a novel mechanism of action (MOA) as an immune-activating anticancer agent. Herein, we describe new 2-(ortho-substituted benzyl)-indole derivatives as RORγ agonists by scaffold hopping based on clinical RORγ antagonist VTP-43742. Interestingly, subtle structural differences of the compounds led to the opposite biological MOA. After rational optimization for structure-activity relationship and pharmacokinetic profile, we identified a potent RORγ agonist compound 17 that was able to induce the production of IL-17 and IFNγ in tumor tissues and elicit antitumor efficacy in MC38 syngeneic mouse colorectal tumor model. This is the first comprehensive work to demonstrate the in vivo antitumor efficacy of an RORγ agonist.

Super-resolution of Pneumocystis carinii pneumonia CT via self-attention GAN.

BACKGROUND AND OBJECTIVE: Computed tomography (CT) examination plays an important role in screening suspected and confirmed patients in pneumocystis carinii pneumonia (PCP), and the efficient acquisition of high-quality medical CT images is essential for the clinical application of computer-aided diagnosis technology. Therefore, improving the resolution of CT images of pneumonia is a very important task. METHODS: Aiming at the problem of how to recover the texture details of the reconstructed PCP CT super-resolution image, we propose the image super-resolution reconstruction model based on self-attention generation adversarial network (SAGAN). In the SAGAN algorithm, a generator based on self-attention mechanism and residual module is used to transform a low-resolution image into a super-resolution image. A discriminator based on depth convolution network tries to distinguish the difference between the reconstructed super-resolution image and the real super-resolution image. In terms of loss function construction, on the one hand, the Charbonnier content loss function is used to improve the accuracy of image reconstruction, and on the other hand, the feature value before activation of the pre-trained VGGNet is used to calculate the perceptual loss to achieve accurate texture detail reconstruction of super-resolution images. RESULTS: Experimental results show that our SAGAN algorithm is superior to other state-of-the-art algorithms in both peak signal-to-noise ratio (PSNR) and structural similarity score (SSIM). Specifically, our SAGAN method can obtain 31.94 dB which is 1.53 dB better than SRGAN on Set5 dataset for 4 enlargements. CONCLUSION: Our SAGAN method can reconstruct more realistic PCP CT images with clear texture, which can help experts diagnose the condition of PCP.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

INTRODUCTION: CD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation. METHODS: CD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model. RESULTS: SHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts. CONCLUSION: Our findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.