c-Cbl-mediated neddylation antagonizes ubiquitination and degradation of the TGF-ß type II receptor.

Transforming growth factor ß (TGF-ß) is a potent antiproliferative factor in multiple types of cells. Deregulation of TGF-ß signaling is associated with the development of many cancers, including leukemia, though the molecular mechanisms are largely unclear. Here, we show that Casitas B-lineage lymphoma (c-Cbl), a known proto-oncogene encoding an ubiquitin E3 ligase, promotes TGF-ß signaling by neddylating and stabilizing the type II receptor (TßRII). Knockout of c-Cbl decreases the TßRII protein level and desensitizes hematopoietic stem or progenitor cells to TGF-ß stimulation, while c-Cbl overexpression stabilizes TßRII and sensitizes leukemia cells to TGF-ß. c-Cbl conjugates neural precursor cell-expressed, developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, to TßRII at Lys556 and Lys567. Neddylation of TßRII promotes its endocytosis to EEA1-positive early endosomes while preventing its endocytosis to caveolin-positive compartments, therefore inhibiting TßRII ubiquitination and degradation. We have also identified a neddylation-activity-defective c-Cbl mutation from leukemia patients, implying a link between aberrant TßRII neddylation and leukemia development.

6'-ß-Fluoro-Homoaristeromycin and 6'-Fluoro-Homoneplanocin A Are Potent Inhibitors of Chikungunya Virus Replication through Their Direct Effect on Viral Nonstructural Protein 1.

Alphaviruses are arthropod-borne, positive-stranded RNA viruses capable of causing severe disease with high morbidity. Chikungunya virus (CHIKV) is an alphavirus that causes a febrile illness which can progress into chronic arthralgia. The current lack of vaccines and specific treatment for CHIKV infection underscores the need to develop new therapeutic interventions. To discover new antiviral agents, we performed a compound screen in cell culture-based infection models and identified two carbocyclic adenosine analogues, 6'-ß-fluoro-homoaristeromycin (FHA) and 6'-fluoro-homoneplanocin A (FHNA), that displayed potent activity against CHIKV and Semliki Forest virus (SFV) with 50% effective concentrations in the nanomolar range at nontoxic concentrations. The compounds, designed as inhibitors of the host enzyme S-adenosylhomocysteine (SAH) hydrolase, impeded postentry steps in CHIKV and SFV replication. Selection of FHNA-resistant mutants and reverse genetics studies demonstrated that the combination of mutations G230R and K299E in CHIKV nonstructural protein 1 (nsP1) conferred resistance to the compounds. Enzymatic assays with purified wild-type (wt) SFV nsP1 suggested that an oxidized (3'-keto) form, rather than FHNA itself, directly inhibited the MTase activity, while a mutant protein with the K231R and K299E substitutions was insensitive to the compound. Both wt nsP1 and the resistant mutant were equally sensitive to the inhibitory effect of SAH. Our combined data suggest that FHA and FHNA inhibit CHIKV and SFV replication by directly targeting the MTase activity of nsP1, rather than through an indirect effect on host SAH hydrolase. The high potency and selectivity of these novel alphavirus mRNA capping inhibitors warrant further preclinical investigation of these compounds.

Neddylation pathway regulates the proliferation and survival of macrophages.

Neddylation is a new type of protein post-translational modification which adds the ubiquitin-like molecule Nedd8 to target proteins. The well-identified targets of neddylation are cullins, which serve as essential components of Cullin-RING E3 ligases (CRL). It is reported that inhibition of neddylation repressed NF-κB-mediated proinflammatory cytokine production in macrophages. However, the role of neddylation in the proliferation and survival of macrophages has not been well defined. Here we report that partial inactivation of the neddylation pathway by a specific Nedd8-activating enzyme E1 (NAE) inhibitor MLN4924 reduced LPS-induced production of the proinflammatory cytokines TNF-α and IL-6 without obvious impairment of cell viability. However, persistent and severe inactivation of neddylation by MLN4924 significantly inhibited cell proliferation by inducing G2 phase cell-cycle arrest and further triggered cell death by inducing apoptosis in RAW264.7 macrophages. Mechanistic analysis revealed that inactivation of neddylation blocked cullin neddylation, inhibited CRL E3 ligase activity, and thus led to the accumulation of CRL substrates, resulting in cell-cycle arrest, DNA damage response and apoptosis. The findings revealed that neddylation serves as an important signaling pathway regulating the proliferation and survival of macrophages.

New RNA purine building blocks, 4'-selenopurine nucleosides: first synthesis and unusual mixture of sugar puckerings.

Writer's blocks: The first synthesis of RNA purine building blocks, 4'-selenoadenosine and 4'-selenoguanosine was achieved from D-ribose by regioisomeric rearrangement, which was confirmed by X-ray crystallography. 4'-Selenoadenosine exists in an unusual mixture of north and south conformers in the solid state.

In Silico Discovery of 5'-Modified 7-Deoxy-7-ethynyl-4'-thioadenosine as a HASPIN Inhibitor and Its Synergistic Anticancer Effect with the PLK1 Inhibitor.

Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4'-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors. In silico apporoaches utilizing transcriptomic and chemical similarities with known compounds and KINOMEscan profiles unveiled the HASPIN inhibitor LJ4827. LJ4827's specificity and potency as a HASPIN inhibitor were verified through in vitro kinase assay and X-ray crystallography. HASPIN inhibition by LJ4827 reduced histone H3 phosphorylation and impeded Aurora B recruitment in cancer cell centromeres but not in noncancer cells. Through transcriptome analysis of lung cancer patients, PLK1 was determined as a druggable synergistic partner to complement HASPIN inhibition. Chemical or genetic PLK1 perturbation with LJ4827 effectuated pronounced lung cancer cytotoxicity in vitro and in vivo. Therefore, LJ4827 is a novel anticancer therapeutic for selectively impeding cancer mitosis through potent HASPIN inhibition, and simultaneous HASPIN and PLK1 interference is a promising therapeutic strategy for lung cancer.

Structure-Activity Relationships of Truncated 1'-Homologated Carbaadenosine Derivatives as New PPARγ/δ Ligands: A Study on Sugar Puckering Affecting Binding to PPARs.

Peroxisome proliferator-activated receptors (PPARs) are associated with the regulation of metabolic homeostasis. Based on a previous report that 1'-homologated 4'-thionucleoside acts as a dual PPARγ/δ modulator, carbocyclic nucleosides 2-5 with various sugar conformations were synthesized to determine whether sugar puckering affects binding to PPARs. (S)-conformer 2 was synthesized using Charette asymmetric cyclopropanation, whereas (N)-conformer 3 was synthesized using stereoselective Simmons-Smith cyclopropanation. All synthesized nucleosides did not exhibit binding affinity to PPARα but exhibited significant binding affinities to PPARγ/δ. The binding affinity of final nucleosides to PPARγ did not differ significantly based on their conformation, but their affinity to PPARδ depended greatly on their conformation, correlated with adiponectin production. (N)-conformer 3h was discovered to be the most potent PPARδ antagonist with good adiponectin production, which exhibited the most effective activity in inhibiting the mRNA levels of LPS-induced IL-1ß expression in RAW 264.7 macrophages, implicating its anti-inflammatory activity.

A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction in vitro and in vivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A2A/A3 Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity.

Based on hA2AAR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2AAR agonists into antagonists while maintaining affinity toward hA3AR. The final compounds of 2,8-disubstituted-N6-substituted 4'-thionucleosides, or 4'-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA2AAR, including 5d with the highest affinity (Ki,A2A = 7.7 ± 0.5 nM). The hA2AAR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA3AR. Structural SAR features and docking studies supported different binding modes at A2AAR and A3AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A2AAR/A3AR nucleoside antagonists would be promising drug candidates for immune-oncology.

A3 adenosine receptor agonist reduces brain ischemic injury and inhibits inflammatory cell migration in rats.

A3 adenosine receptor (A3AR) is recognized as a novel therapeutic target for ischemic injury; however, the mechanism underlying anti-ischemic protection by the A3AR agonist remains unclear. Here, we report that 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyl-4'-thioadenosine (LJ529), a selective A3AR agonist, reduces inflammatory responses that may contribute to ischemic cerebral injury. Postischemic treatment with LJ529 markedly reduced cerebral ischemic injury caused by 1.5-hour middle cerebral artery occlusion, followed by 24-hour reperfusion in rats. This effect was abolished by the simultaneous administration of the A3AR antagonist MRS1523, but not the A2AAR antagonist SCH58261. LJ529 prevented the infiltration/migration of microglia and monocytes occurring after middle cerebral artery occlusion and reperfusion, and also after injection of lipopolysaccharides into the corpus callosum. The reduced migration of microglia by LJ529 could be related with direct inhibition of chemotaxis and down-regulation of spatiotemporal expression of Rho GTPases (including Rac, Cdc42, and Rho), rather than by biologically relevant inhibition of inflammatory cytokine/chemokine release (eg, IL-1ß, TNF-α, and MCP-1) or by direct inhibition of excitotoxicity/oxidative stress (not affected by LJ529). The present findings indicate that postischemic activation of A3AR and the resultant reduction of inflammatory response should provide a promising therapeutic strategy for the treatment of ischemic stroke.

Anticancer activity of IRAK-4 inhibitors against canine lymphoid malignancies.

The interleukin-1 receptor-related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signalling seen in some B-cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B-cell subtype of human diffuse large B-Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK-4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL-1 and CL-1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL-1 and CL-1 cells were subjected to concentration- and time-dependent treatment with an IRAK-4 inhibitor and assessed for viability, TLR signalling association and apoptosis using a cell counting Kit-8 assay, Western blotting and flow cytometry. The GL-1 and CL-1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK-4 inhibitor reduced cell viability in a dose- and time-dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF-κB and STAT3, and induced apoptosis in GL-1 and CL-1 cells. The anticancer effect of the IRAK-4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling.