Structural Modification and Biological Evaluation of 2,8-Disubstituted Adenine and Its Nucleosides as A2A Adenosine Receptor Antagonists: Exploring the Roles of Ribose at Adenosine Receptors.

Building on the preceding structural analysis and a structure-activity relationship (SAR) of 8-aryl-2-hexynyl nucleoside hA2AAR antagonist 2a, we strategically inverted C2/C8 substituents and eliminated the ribose moiety. These modifications aimed to mitigate potential steric interactions between ribose and adenosine receptors. The SAR findings indicated that such inversions significantly modulated hA3AR binding affinities depending on the type of ribose, whereas removal of ribose altered the functional efficacy via hA2AAR. Among the synthesized derivatives, 2-aryl-8-hexynyl adenine 4a demonstrated the highest selectivity for hA2AAR (Ki,hA2A = 5.0 ± 0.5 nM, Ki,hA3/Ki,hA2A = 86) and effectively blocked cAMP production and restored IL-2 secretion in PBMCs. Favorable pharmacokinetic properties and a notable enhancement of anticancer effects in combination with an mAb immune checkpoint blockade were observed upon oral administration of 4a. These findings establish 4a as a viable immune-oncology therapeutic candidate.

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.

Chemical Probe to Identify the Cellular Targets of the Reactive Lipid Metabolite 2- trans-Hexadecenal.

Lipid-derived electrophiles (LDEs) are reactive metabolites, which can covalently modify proteins and DNA and regulate diverse cellular processes. 2- trans-Hexadecenal (2-HD) is a byproduct of sphingolipid metabolism, involved in cytoskeletal reorganization, DNA damage, and apoptosis. In addition, the loss of ALDH3A2, an enzyme removing 2-HD in cells, is responsible for Sjörgen-Larsson Syndrome (SJS), suggesting that accumulation of 2-HD could lead to pathogenesis. However, the targets and the precise mechanisms of 2-HD are not well characterized. Herein, we report an alkyne-2-HD derivative as a bioorthogonal probe to explore the functions of 2-HD. We identified more than 500 potential cellular targets. Among them, the pro-apoptotic protein Bax can be covalently modified by 2-HD directly at the conserved Cys62 residue. Our work provided new chemical tools to explore the cellular functions of LDEs and revealed new mechanistic insights of the deregulation of lipid metabolism in diseases.

p300-mediated acetylation increased the protein stability of HIPK2 and enhanced its tumor suppressor function.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in development and tumor suppression. One of the prominent features of this kinase is that it is tightly regulated by proteasomal degradation. In the present study, we present evidence suggesting that the protein stability of HIPK2 can be regulated by p300-mediated acetylation. p300 increased the protein level of HIPK2 via its acetyltransferase activity. p300 increased the acetylation of HIPK2 while decreased polyubiquitination and its proteasomal degradation. We also observed that DNA damage induced acetylation of HIPK2 along with an increase in the protein amount, which was inhibited by p300 RNAi. Importantly, p300 promoted p53 activation and the HIPK2-mediated suppression of cell proliferation, suggesting acetylation-induced HIPK2 stabilization contributed to the enhanced activation of HIPK2. Overexpression of p300 promoted the HIPK2-mediated suppression of tumor growth in mouse xenograft model as well. Taken together, our data suggest that p300-mediated acetylation of HIPK2 increases the protein stability of HIPK2 and enhances its tumor suppressor function.

PARP1 regulates the protein stability and proapoptotic function of HIPK2.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2.

SIRT1 negatively regulates the protein stability of HIPK2.

In the present study, we investigated whether a histone deacetylase sirtuin 1 (SIRT1) can regulate the protein stability of homeodomain-interacting protein kinase 2 (HIPK2). We observed the evidence of molecular interaction between SIRT1 and HIPK2. Interestingly, overexpression or pharmacological activation of SIRT1 promoted ubiquitination and the proteasomal degradation of HIPK2 whereas inhibition of SIRT1 activity increased the protein level of HIPK2. Furthermore, a SIRT1 activator decreased the level of HIPK2 acetylation whereas an inhibitor increased the acetylation level. These results suggest that SIRT1 may deacetylate and promote the ubiquitination and subsequent proteasomal degradation of HIPK2.

Resveratrol upregulated heat shock proteins and extended the survival of G93A-SOD1 mice.

In the present study, we investigated whether resveratrol, a SIRT1 activator, can suppress the motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis. Chronic intraperitoneal injection of resveratrol delayed the disease onset and extended survival of the transgenic mice overexpressing G93A-SOD1. The number of surviving motor neurons increased in the resveratrol-injected G93A mice. Importantly, the levels of Hsp25 and Hsp70 were elevated while the level of heat shock factor 1 (HSF1) acetylation decreased in the spinal cords of the resveratrol-injected G93A mice. Our data suggest that resveratrol may protect motor neurons from the mutant SOD1-induced neurotoxicity by promoting SIRT1-mediated deacetylation of HSF1 and subsequent upregulation of Hsps.

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11.

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.