Background: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effects against heart failure in patients with diabetes, and there is intense interest to identify the underlying molecular mechanisms that afford this protection. Because the induction of the late component of the cardiac sodium channel current (late- I _Na ) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late- I _Na . Methods: Electrophysiological, in silico molecular docking, molecular, calcium imaging, and whole heart perfusion techniques were used to address this question. Results: The SGLT2 inhibitor empagliflozin reduced late- I _Na in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the long QT syndrome 3 mutations R1623Q or ΔKPQ. Empagliflozin, dapagliflozin, and canagliflozin are all potent and selective inhibitors of H ₂ O ₂ -induced late- I _Na (half maximal inhibitory concentration = 0.79, 0.58, and 1.26 µM, respectively) with little effect on peak sodium current. In mouse cardiomyocytes, empagliflozin reduced the incidence of spontaneous calcium transients induced by the late- I _Na activator veratridine in a similar manner to tetrodotoxin, ranolazine, and lidocaine. The putative binding sites for empagliflozin within Nav1.5 were investigated by simulations of empagliflozin docking to a three-dimensional homology model of human Nav1.5 and point mutagenic approaches. Our results indicate that empagliflozin binds to Nav1.5 in the same region as local anesthetics and ranolazine. In an acute model of myocardial injury, perfusion of isolated mouse hearts with empagliflozin or tetrodotoxin prevented activation of the cardiac NLRP3 (nuclear-binding domain-like receptor 3) inflammasome and improved functional recovery after ischemia. Conclusions: Our results provide evidence that late- I _Na may be an important molecular target in the heart for the SGLT2 inhibitors, contributing to their unexpected cardioprotective effects.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses. Infection with influenza viruses is associated with high morbidity and mortality. Therefore, vaccination is recommended in immunosuppressed patients, however often the post-vaccine induced protection is insufficient. Factors associated with reduced vaccine responses may guide preventive strategies and could offer novel targets for adjuvants. Here, we explore the impact of IL-28B on B- and T-cell responses during vaccination. We found that a single nucleotide polymorphism (minor allele genotype) in the IL-28B gene was associated with a significant increase in the antibody seroconversion rate following influenza vaccination. Interestingly, this SNP reduces the expression of IL-28B. In addition, in vitro stimulation of peripheral blood mononuclear cells from patients with the SNPs had increased IL-4 production in CD4 T-cells. As a potential mechanism, we show that recombinant IL-28B inhibits influenza stimulated Th2 cytokine release, B-cell activation/proliferation and H1N1-induced IgG secretion. Next, we developed antagonistic peptides to block the IFN-λ receptor. Pre-treatment with the antagonistic peptides increased in vitro B-cell activation and antibody production in healthy individuals and transplant recipients. Together, these findings identify IL-28B as a key regulator of Th1/Th2 balance during influenza vaccination. Blockade of the IFN-λ receptor with antagonistic peptides may offer a novel strategy to augment vaccine responses.