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.

PDF file - 1MB, MLN4924 induced more apoptosis in MEF-Atg7-KO compared to MEF-Atg7-WT

PDF file - 924K, No obvious dysfunction of liver, kidney or muscle of animals was observed post MLN4924 treatment

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PDF file - 979K, MLN4924-induced autophagy was partially rescued by N-Acetyl-L-cysteine (NAC)

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.

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.

<p>PDF file - 1.2MB, Induction of cell cycle arrest and apoptosis by MLN4924 in Hep G2 cells</p>

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-1β expression in RAW 264.7 macrophages, implicating its anti-inflammatory activity.

HASPIN, a mitotic kinase to phosphorylate Histone H3 at centromere, has been studied as a promising target for anti-cancer therapy. However, as HASPIN belongs to atypical kinase, lacking the Asp-Phe-Gly (DFG) motif, development from the chemical library of kinase inhibitors with conventional pharmacophore, would be technically challenging. In particular, one of adenosine analogues (LJ4827), a potent inhibitor of multi-kinases, showing clear anti-cancer activity in vitro and in vivo, was predicted as a HASPIN inhibitor by computation analysis of its transcriptome profile in cancer cells to drug-omic data set in the connectivity MAP (cMAP) as it shared similar transcriptome profile of 5-iodotubercidin (5ITU). The specificity and potency as HASPIN inhibitor of LJ4827 (IC50 = 0.45 nM) validated by in vitro kinase screening and consequent 3D structure modeling. As expected, treatment of LJ4827 in cancer cell lines efficiently delayed mitotic progression without double strand break (DSB) unlike 5ITU and significantly attenuated Aurora B localization at centromere. Along with clinical significance of HASPIN expression in lung cancer patients, mitotic gene signature closely associated to high expression of HASPIN, revealed the poor prognosis. Additional computational analysis of kinase perturbation data to predict the dependency of mitotic kinase in the absence of HASPIN activity, revealed the synthetic lethal effect of cotreatment of the chemical inhibitor of BUB1, PLK1 or AURKA with LJ4827. These data suggest that combined inhibition of HASPIN with the novel inhibitor and key mitotic kinases for centromere/kinetochore regulation would be effectivity therapeutic approach for cancer therapy. Citation Format: Eun-Ji Kwon, Karishma Mashelkar, Hyuk Woo Lee, Yoon-Ze Shin, Lak Shin Jeong, Hyuk-Jin Cha. Identification of a novel HASPIN inhibitor and the synthetic lethal partner by computational analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3949.

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.

The development of anticancer drugs remains challenging owing to the potential for drug resistance. The simultaneous inhibition of multiple targets involved in cancer could overcome resistance, and these agents would exhibit higher potency than single-target inhibitors. Protein kinases represent a promising target for the development of anticancer agents. As most multi-kinase inhibitors are heterocycles occupying only the hinge and hydrophobic region in the ATP binding site, we aimed to design multi-kinase inhibitors that would occupy the ribose pocket, along with the hinge and hydrophobic region, based on ATP-kinase interactions. Herein, we report the discovery of a novel 4′-thionucleoside template as a multi-kinase inhibitor with potent anticancer activity. The in vitro evaluation revealed a lead 1g (7-acetylene-7-deaza-4′-thioadenosine) with potent anticancer activity, and marked inhibition of TRKA, CK1δ, and DYRK1A/1B kinases in the kinome scan assay. We believe that these findings will pave the way for developing anticancer drugs.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6′,6′-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5’ end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a ∼100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication. Importance Currently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.

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′-β-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.

Following our report that A3 adenosine receptor (AR) antagonist 1 exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1'-homologated adenosine analogues 4a-4t, as dual PPARγ/δ modulators without AR binding. Removal of binding affinity to A3AR was achieved by 1'-homologation, and PPARγ/δ dual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγ affinity, indicating that the C2 position defines a pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.

Study objectivesNon‐alcoholic fatty liver disease (NAFLD) has been considered leading cause of developing liver failure. However, the underlying mechanisms for transition from benign steatosis to clinically significant non‐alcoholic steatohepatitis (NASH) and non‐alcoholic steatofibrosis (NASF) have not been fully understood.MethodIn mouse model, NAFLD was induced by fast food diet (FFD) with a high glucose‐fructose solution for 24 weeks. For secretome proteomics, we used primary hepatocytes (HPs) isolated from normal chow diet (NCD) or FFD‐fed liver. The biased agonist of A3 adenosine receptor (A3AR), FM101 was utilized for the study of A3AR signaling.ResultsIn secretome proteomics, we found α1‐antitrypsin (A1AT) was decreased 0.2‐fold in FFD hepatocytes compared with NCD hepatocytes. Consistently, serum levels of A1AT from mice and human patients with NAFLD were significantly decreased compared to normal group. Conversely, the mRNA and protein level of Neutrophil Elastase (NE) were increased in FFD‐fed liver compared to NCD‐fed liver. We next confirmed inflammatory role of NE in Kupffer cells (KCs) as various cytokines (CCL2, TNF‐α, IL‐1β, iNOS and IL‐6) were increased in KCs with treatment of NE. Of note, NE has been reported to exert proteolytic activity that can prevent activation of G‐protein‐coupled receptors (GPCRs). We observed similar proteolysis that A3AR, a member of the adenosine receptor group of GPCRs, was cleaved in hepatic stellate cells (HSCs) after treatment of NE, indicating that A1AT‐NE system may regulate the A3AR signaling. Interestingly, A3AR was predominantly expressed in KCs and HSCs, but not in HPs. In vitro activation of A3AR signaling with FM101 significantly inhibited activation of KCs and HSCs as demonstrated by inflammatory cytokines (TNF‐α, IL‐6 and IL‐1β), and profibrogenic genes (Col1a1, Timp1 Acta2) expression, respectively. Moreover, in vivo activation of A3AR with FM101, lowered liver weight, liver to body ratio, and serum level of ALT and cholesterol induced by FFD. Furthermore, FM101 effectively improved steatofibrosis as assessed by quantification of collagen deposition. Mechanistically, FM101 treatment strongly blocked mitochondrial oxidative phosphorylation. Using pMito‐Timer and Mt‐Keima system, we observed FM101‐induced mitochondria vulnerability as demonstrated by increased accumulation of damaged mitochondria and subsequence mitophagic degradation. Consequently, CCCP‐induced cell death in macrophage was further enhanced by FM101 treatment.ConclusionsThe imbalance between A1AT and NE in simple steatosis mediates the progression of NASH and NASF through proteolytic inactivation of A3AR in KCs and HSCs. Thus, A3AR agonism with FM101 may have therapeutic potentials for treatment of NAFLD.Support or Funding InformationNational Research Foundation of Korea (NRF) ‐ 2019R1A2C1090178FM101 ameliorates FFD‐induced steatohepatitis and stetofibrosisFigure 1Adenosine A3 receptor signaling contributes to mitochondria vulnerabilityFigure 2

The 6'-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6'-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N6-methyladenosine analogues 2a-e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6',6'-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6'-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.

Truncated 4′-thionucleosides 1–4 and 4′-oxonucleosides 5–8 as potent and selective A3AR antagonists were synthesized from d-mannose and d-erythronic acid γ-lactone, respectively. These nucleosides were evaluated for their anti-fibrotic renoprotective activity in TGF-β1-treated murine proximal tubular (mProx) cells. Their antagonistic activities for A3AR were proportional to their inhibitory activities against TGF-β1-induced collagen I upregulation in mProx cells. This result suggests that the binding affinity of A3AR antagonists is closely correlated with their anti-fibrotic activity. Thus, A3AR antagonists might be novel therapeutic candidates for treating chronic kidney disease.