Rigidified nucleoside derivatives with (N)-methanocarba replacement of ribose have been repurposed as peripheral subtype-selective 5-HT2B serotonin receptor antagonists for heart and lung fibrosis and intestinal/vascular conditions. 4 '-Cyano derivative 40 (MRS8209; Ki , 4.27 nM) was 47-fold (human binding, but not rat and mouse) and 724-fold (functionally) selective at 5-HT2BR, compared to antitarget 5-HT2CR, and predicted to form a stable receptor complex using docking and molecular dynamics. 4 '-Cyano substituents enhanced 5-HT2BR affinity (typically 4-5-fold compared to 4 '-CH2OH), depending on an N 6 group larger than methyl. Asymmetric N 6 groups (4 '-cyano-2-halo derivatives 33-35 and 37) provided potent 5-HT2BR Ki values (7-22 nM). A 4 '-CH2CN substituent was less effective than 4 '-CN at increasing 5-HT2BR affinity, while a 4 '-CHF2 group produced high 5-HT2B affinity/selectivity. A 2-benzylthio-adenine group with unsubstituted 6-NH2 shifted the typical selectivity pattern toward potent 5-HT2C binding. Thus, the SAR suggests that N 6-cyclopentyl-4 '-cyano modifications are promising, with an interdependence among the substituent positions.

2-Arylethynyl (N)-methanocarba adenosine 5 '-methylamides are selective A(3) adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9-11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A(3)AR K-i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A(3)AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4 '-truncated derivatives had greatly reduced hA(3)AR affinity, even containing affinity-enhancing N-6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A(3)AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA(3)AR miniG alpha(i) recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a beta-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E-max) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A(3)AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a-3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A(3)AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A(3)AR agonist SAR for (N)-methanocarba adenosines.

(N)-Methanocarba adenosine derivatives were structurally modified to target 5-HT2B serotonin receptors as antagonists, predominantly containing branched N6-alkyl groups. N6-Dicycloalkyl-methyl groups, including their asymmetric variations, as well as 2-iodo, were found to generally favor 5-HT2Rs, while only N6-dicyclohexyl-methyl derivative 35 showed weak 5-HT2AR affinity (Ki 3.6 μM). The highest 5-HT2BR affinities were Ki 11-23 nM (N6-dicyclopropyl-methyl-2-iodo 11, 2-chloro-5'-deoxy-5'-methylthio 15 and N6-((R)-cyclobuty-cyclopropyl-methyl)-2-iodo 43), and Ki 73 nM at 5-HT2CR for 36. Direct comparison of adenine ribosides and their corresponding rigid (N)-methanocarba derivatives (cf. 51 and MRS8099 45) indicated a multifold affinity enhancement with the bicyclic ring system. Compounds 43, 45 and 48 were functional 5-HT2BR (KB 2-3 nM) and 5-HT2CR (KB 79-328 nM) antagonists in a Gq-mediated calcium flux assay, with 5-HT2BR functional selectivity ranging from 45- (48) to 113-fold (43). Substantial adenosine receptor (AR) affinity (Ki, A1AR < Ki, A3AR < Ki, A2AAR) was still present in this series, suggestive of dual acting compounds: 5-HT2B antagonist and A1AR agonist, potentially useful for treating chronic conditions (fibrosis; pain). Given its affinity (17 nM) and moderate 5-HT2BR binding selectivity (32-fold vs. 5-HT2CR, 4-fold vs. A1AR), 43 (MRS7925) could potentially be useful for anti-fibrotic therapy.

(N)-Methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexane replacing furanose) were chain-extended at N6 and C2 positions with terminal alkenes for ring closure. The resulting macrocycles of 17-20 atoms retained affinity, indicating a spatially proximal orientation of these receptor-bound chains, consistent with molecular modeling of 12. C2-Arylethynyl-linked macrocycle 19 was more A3AR-selective than 2-ether-linked macrocycle 12 (both 5'-methylamides, human (h) A3AR affinities (Ki): 22.1 and 25.8 nM, respectively), with lower mouse A3AR affinities. Functional hA3AR comparison of two sets of open/closed analogues in β-arrestin2 and Gi/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gαi isoforms were slightly less than its hA3AR binding affinity (Ki: 1.4 nM), while the Gαi1 and Gαi2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gαi2-coupling was enhanced in macrocycle 12 (EC50 2.56 nM, ∼40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the Gαi1 potency by 7.5-fold. The macrocycles 12 and 19 and their open precursors 11 and 18 potently stimulated β-arrestin2 recruitment, with EC50 values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to 1. This example of macrocyclization altering the coupling pathways of small-molecule (nonpeptide) GPCR agonists is the first for potent and selective macrocyclic AR agonists. These initial macrocyclic derivatives can serve as a guide for the future design of macrocyclic AR agonists displaying unanticipated pharmacology.

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

Abstract Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

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BACKGROUND:Mouse models are highly effective for studying the pathophysiology of lung adenocarcinoma and evaluating new treatment strategies. Treatment efficacy is primarily determined by the total tumor burden measured on excised tumor specimens. The measurement process is time-consuming and prone to human errors. To address this issue, we developed a novel deep learning model to segment lung tumor foci on digitally scanned hematoxylin and eosin (H&E) histology slides.METHODS:Digital slides of 239 mice from 9 experimental cohorts were split into training (n=137), validation (n=37), and testing cohorts (n=65). Image patches of 500×500 pixels were extracted from 5× and 10× magnifications, along with binary masks of expert annotations representing ground-truth tumor regions. Deep learning models utilizing DeepLabV3+ and UNet architectures were trained for binary segmentation of tumor foci under varying stain normalization conditions. The performance of algorithm segmentation was assessed by Dice Coefficient, and detection was evaluated by sensitivity and positive-predictive value (PPV).RESULTS:The best model on patch-based validation was DeepLabV3+ using a Resnet-50 backbone, which achieved Dice 0.890 and 0.873 on validation and testing cohort, respectively. This result corresponded to 91.3 Sensitivity and 51.0 PPV in the validation cohort and 93.7 Sensitivity and 51.4 PPV in the testing cohort. False positives could be reduced 10-fold with thresholding artificial intelligence (AI) predicted output by area, without negative impact on Dice Coefficient. Evaluation at various stain normalization strategies did not demonstrate improvement from the baseline model.CONCLUSIONS:A robust AI-based algorithm for detecting and segmenting lung tumor foci in the pre-clinical mouse models was developed. The output of this algorithm is compatible with open-source software that researchers commonly use.

The A3 adenosine receptor (A3AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure-activity relationship of a previously reported A3AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [3H]MRS7799. A3AR Kd values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.

Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

Commensal microbiota has emerged as an essential biomarker and regulator of both tumorigenesis and response to cancer therapy. However, our current knowledge about microbiota in cancer has been largely limited to intestinal microbiota. As a mucosal organ harboring one of the largest surface areas in the body, the lung is exposed to a variety of microbes through inhalation and micro-aspiration, and is colonized by a diverse bacterial community in both physiological and pathological conditions. Importantly, increasing evidence has linked the lung microbiome to cancer development. Studies in lung cancer patients and mouse models have revealed tumor-associated dysregulation of the local microbiome in the lung, which in turn impacts cancer progression by shaping the tumor microenvironment and modulating the activity of tumor-infiltrating immune cells. These findings not only provide novel mechanistic insight into the biology of lung cancer but also shed light on new therapeutic targets and strategies for lung cancer prevention and treatment. The goal of this review is to discuss the key findings, remaining questions, and future directions in this new and exciting field.

In recent years, myeloid-derived suppressor cells (MDSC) have emerged as one of the major inhibitors of immune effector cell function in cancer. MDSC represent a heterogeneous population of largely immature myeloid cells that are characterized by a pathological state of activation and display potent immune suppressive activity. Two major subsets of MDSC have been identified: monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC). PMN-MSDC share phenotypic and morphologic features with neutrophils, whereas M-MDSC are similar to monocytes and are characterized by high plasticity. Differentiation of M-MDSC to macrophages and dendritic cells is shaped by tumor microenvironment. In recent years, the mechanisms of this process start to emerge.

The mammary gland undergoes fast cell proliferation during early pregnancy, yet the mechanism to ensure genome integrity during this highly proliferative stage is largely unknown. We show that pregnancy triggers replicative stresses leading to genetic instability in mice carrying a mammary specific disruption of breast cancer associated gene-1 (BRCA1). The fast cell proliferation was correlated with enhanced expression of most genes encoding replisomes, which are positively regulated by estrogen/ERα signaling but negatively regulated by BRCA1. Our further analysis revealed two parallel signaling pathways, which are mediated by ATR-CHK1 and WEE1-MCM2 and are responsible for regulating DNA replication checkpoint. Upon DNA damage, BRCA1 deficiency markedly enhances DNA replication initiation and preferably impairs DNA replication checkpoint mediated by ATR and CHK1. Meanwhile, DNA damage also activates WEE1-MCM2 signaling, which inhibits DNA replication initiation and enables BRCA1-deficient cells to avoid further genomic instability. Finally, we demonstrated that overriding this defense by WEE1 inhibition in combination with cisplatin, which causes DNA damage, serves as a promising therapeutic approach for killing BRCA1-deficient cancer cells.

HIV type 1 (HIV-1) replicates preferentially in IL-4-producing CD4 T cells for unclear reasons. We show increased HIV-1 expression is irrespective of viral tropism for chemokine receptors as previously suggested, but rather transcription of the HIV-1 long terminal repeat (LTR) is increased in IL-4-producing CD4 T cells. Increased expression of HIV-1 message is also confirmed in IL-4-producing CD4 T cells from HIV-1-infected individuals ex vivo. In exploring a transcriptional mechanism, we identify a novel c-maf (required for IL-4 expression) transcription factor binding site just upstream of the dual NF-κB/NFAT binding sites in the proximal HIV-1 LTR. We demonstrate that c-maf binds this site in vivo and synergistically augments HIV-1 transcription in cooperation with NFAT2 and NF-κB p65, but not NFAT1 or NF-κB p50. Conversely, small interfering RNA inhibition of c-maf reduces HIV-1 transcription in IL-4-producing T cells. Thus, c-maf increases HIV-1 expression in IL-4-producing CD4 T cells by binding the proximal HIV-1 LTR and augmenting HIV-1 transcription in partnership with NFAT2 and NF-κB p65 specifically. This has important implications for selective targeting of transcription factors during HIV-1 infection because, over the course of HIV-1 progression/AIDS, IL-4-producing T cells frequently predominate and substantially contribute to disease pathology.

The human immunodeficiency virus, HIV-1, infects human CD4 T cells, including Th1 and Th2 helper cell subsets. The virus relies on host transcription factors, such as NFkB and NFAT for its transcription and expression. It has previously been shown that HIV-1 preferentially replicates in Th2 cells, but the mechanism of action for this finding remains unknown. c-maf is a Th2-restricted transcription factor that is critically important for differentiation along a Th2, but not Th1 lineage, and for transcription of the prototypic Th2 cytokine, IL-4. c-maf directly binds the proximal IL-4 promoter and acts synergistically with a neighboring NFAT site. We have demonstrated at the individual cell level that IL-4 positive cells (Th2) preferentially support HIV-1 replication compared to IFNg positive (Th1) cells. In studying the HIV-1 long terminal repeat (LTR)/promoter sequence, we identified a MARE (maf-recognition element) located just proximal (5') to the dual NFkB/NFAT binding sites. We show that the HIV-1 MARE binds recombinant maf protein and abuts NFAT binding as detected by DNase I in vitro footprinting. In addition, this HIV-1 MARE demonstrates identical mobility shifts compared to the IL-4 promoter MARE in gel-shift assays using nuclear extracts from activated primary human CD4 T cells. Using chromatin immunoprecipitation, we further show that c-maf binds to the HIV-1 LTR in vivo in HIV-1 infected primary human CD4 T cells. Although we have previously shown that both NFAT1 and NFAT2 are capable of transactivating the HIV-1 LTR, we now show for the first time preferential binding in vitro and in vivo of NFAT2 over NFAT1 to the HIV-1 LTR. By comparison, the more abundant NFAT1 family member preferentially binds to the IL-2 promoter. NFAT2 has previously been implicated in Th2 cytokine expression and appears to cooperate with c-maf in binding to the HIV-1 LTR. Functionally, over-expression of cmaf in primary human CD4 T cells cooperatively increases HIV-1 transcription when co-expressed with NFAT1 and 2, and silencing endogenous c-maf expression in primed human CD4 T cells decreases viral transcription. Similarly, over-expression of c-maf alone, or with NFAT1 or 2, increases HIV-1 replication, as measured by intracellular p24/gag expression, in CD4 T cells co-expressing GFP but not in GFP negative/c-maf-negative controls within the same transfected population. Lastly, depletion of c-maf expression by siRNA in primed and HIV-1 infected CD4 T cells decreases p24/gag expression. In summary, the Th2specific transcription factor, c-maf, binds the HIV-1 promoter in cooperation with NFAT transcription factors, primarily NFAT2, to augment HIV-1 transcription and replication in primary human CD4 T cells. These are the first data to mechanistically explain preferential HIV-1 transcription in IL-4 producing (Th2) cells. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005