Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library.

BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.

ENO1 Promotes Lung Cancer Metastasis via HGFR and WNT Signaling-Driven Epithelial-to-Mesenchymal Transition.

ENO1 (α-enolase) expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In this study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of patients with lung cancer. Knockdown of ENO1 decreased cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhanced these processes. Moreover, ENO1 expression promoted tumor growth in orthotopic models and enhanced lung tumor metastasis in tail-vein injection models. These effects were mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-to-mesenchymal transition regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacted with hepatocyte growth factor receptor (HGFR) and activated HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt coreceptor LRP5/6. Activation of these signaling axes decreased GSK3ß activity via Src-PI3K-AKT signaling and inactivation of the ß-catenin destruction complex to ultimately upregulate SLUG and ß-catenin. In addition, we generated a chimeric anti-ENO1 mAb (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuated cancer cell invasion by inhibiting ENO1-mediated GSK3ß inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevented lung tumor metastasis and prolonged survival in animal models. Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and support the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer. SIGNIFICANCE: This study shows that ENO1 promotes lung cancer metastasis via HGFR and WNT signaling and introduces a novel anti-ENO1 antibody for potential therapeutic use in lung cancer.

Prodigiosin-induced cytotoxicity involves RAD51 down-regulation through the JNK and p38 MAPK pathways in human breast carcinoma cell lines.

RAD51 is essential for homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) in mammalian cells. RAD51 is an attractive target for anticancer drugs, given high RAD51 levels are frequently observed in many human tumors and associated with increased resistance to DSBs-inducing chemotherapeutics. Prodigiosin is a bacterial tripyrrole pigment with potent anticancer activity and also provokes DSBs. We hereby aimed to elucidate the role of RAD51 in prodigiosin-induced cytotoxicity. Prodigiosin was found to down-regulate RAD51 in multiple human breast carcinoma cell lines irrespective of p53 status. Mechanistically, prodigiosin lowered RAD51 mRNA expression, whereas blockade of proteasome-mediated degradation failed to restore RAD51 levels following prodigiosin treatment. In addition, prodigiosin triggered phosphorylation of JNK and p38 MAPK, while pharmacological inhibition of JNK or p38 MAPK attenuated prodigiosin-mediated inhibition of RAD51 mRNA expression. Lastly, cells with enforced RAD51 expression showed increased resistance to prodigiosin-induced cytotoxicity as well as inhibition of colony formation. Collectively, we conclude that RAD51 down-regulation represents one of the modes of prodigiosin's cytotoxic action, ostensibly by augmenting the genotoxic effect of prodigiosin through suppression of RAD51-mediated HR repair. Our findings further implicate the use of prodigiosin to potentiate the cytotoxicity of DSB-inducing chemotherapeutics through RAD51 down-regulation.

Prodigiosin down-regulates SKP2 to induce p27(KIP1) stabilization and antiproliferation in human lung adenocarcinoma cells.

BACKGROUND AND PURPOSE: High levels of SKP2 are a poor prognostic factor in multiple human cancers and mostly correlate with low p27(KIP1) levels. Prodigiosin is a bacterial tripyrrole pigment with strong pro-apoptotic activity. Induction of cell cycle blockade underlies one of its anticancer actions but the mechanisms involved are unclear. The aim of this study was to explore the role of the SKP2-p27(KIP1) axis in prodigiosin's cytostatic effect on human lung adenocarcinoma cells. EXPERIMENTAL APPROACH: Prodigiosin's effects on cell cycle progression and long-term cell proliferation of human lung adenocarcinoma cells were characterized by flow cytometry and colony formation assay, respectively. Real-time RT-PCR and promoter activity analyses were performed for assessing transcriptional control, while cycloheximide chase analysis evaluated protein stability. Immunoblotting was employed for mechanistic study. KEY RESULTS: Prodigiosin increased p27(KIP1) expression mainly by stabilizing p27(KIP1) through transcriptional repression of SKP2. Importantly, SKP2 overexpression or p27(KIP1) depletion restored the colony forming capacity of prodigiosin-treated cells. Furthermore, prodigiosin induced PKB dephosphorylation, leading to PKB inhibition as revealed by decreased serine 9 phosphorylation of GSK-3ß. Constitutive PKB activation reduced prodigiosin-induced SKP2 repression. Prodigiosin also down-regulated E2F1 (mediates PI3K/PKB-induced SKP2 transcription), but E2F1 overexpression failed to restore SKP2 expression in prodigiosin-treated cells. CONCLUSIONS AND IMPLICATIONS: Transcriptional repression of SKP2 and the consequent accumulation of p27(KIP1) are essential for prodigiosin's antiproliferative action. Mechanistically, prodigiosin induces PKB inhibition to down-regulate SKP2 in a GSK-3ß- and E2F1-independent manner. Our findings further implicate the potential for developing prodigiosin as a novel class of SKP2-targeting anticancer agent.

1,10-phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme, cytochrome P450 1a1.

1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn(2+), Fe(2+) and Cu(2+) increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn(2+) and Fe(2+) highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.

1-Nitropyrene stabilizes the mRNA of cytochrome P450 1a1, a carcinogen-metabolizing enzyme, via the Akt pathway.

Cytochrome P450 1a1 (Cyp1a1) is a phase I xenobiotic-metabolizing enzyme, the expression of which is mainly driven by the aryl hydrocarbon receptor (AhR). Cyp1a1 messenger (m)RNA is labile. Our study indicates that 1-nitropyrene (1-NP) highly induced Cyp1a1 protein expression, although its induction of AhR transactivation activity was negligible. The fact that the nuclear receptors, CAR, FXR LXR, or PXR, did not induce Cyp1a1 expression indicates that they do not mediate 1-NP's action. When the AhR transcript was degraded by small hairpin (sh)RNA-AhR, 1-NP-induced Cyp1a1 expression largely decreased. In addition, 1-NP did not induce Cyp1a1 in AhR pathway-deficient mutant cells, which indicates that the AhR is essential for 1-NP's action. When Cyp1a1's turnover was examined, 1-NP was able to stabilize the 1-NP- and benzo[a]pyrene (BaP)-induced Cyp1a1 mRNA, but not protein. 1-NP-induced Cyp1a1 mRNA stabilization was mediated by Akt, but not by p38 MAPK, MEK1/2, or JNK. Among aryl hydrocarbons with four annealed phenyl rings, including pyrene, 1-NP, fluoranthene, 3-nitrofluoranthene, chrysene, and 6-nitrochrysene, only 1-NP was able to stabilize Cyp1a1 mRNA. 1-NP's action was gene specific. In conclusion, stabilizing Cyp1a1 mRNA greatly contributed to 1-NP-induced Cyp1a1 expression, which provides new insight into gene regulation by the AhR ligand and mRNA stabilization.

Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines.

Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.

Aldo-keto reductase 1C2 is essential for 1-nitropyrene's but not for benzo[a]pyrene's induction of p53 phosphorylation and apoptosis.

It is reported that diesel exhaust particles contain more 1-nitropyrene (1-NP) than benzo[a]pyrene (B[a]P), both of which are potent carcinogenic compounds. In this study, we show that 1-NP is more potent in reducing cell viability than B[a]P, pyrene, nitrobenzene, and nitromethane. Aldo-keto reductases (AKRs) are enzymes which metabolize polycyclic aromatic hydrocarbons into active metabolites that form PAH-DNA-adducts causing mutagenesis of DNA. We found that the AKR1C2 inhibitor, ursodeoxycholic acid (UA), inhibited 1-NP-induced, but not B[a]P-induced, phosphorylation of p53 and cleavage of poly (ADP-ribose) polymerase (PARP). 1-NP-induced apoptosis was also suppressed by UA, as detected by Hoechst 33342 staining, flow cytometric analysis of subG0/G1 phase and annexin V binding to phosphatidylserine. The AKR1C1 and 1C4 inhibitor, 1,10-phenanthroline (Phen), inhibited the toxic effects of both 1-NP and B[a]P. In contrast, the AKR7A1 and 7A5 inhibitors, succinate and citrate, did not influence the toxic effects of 1-NP or B[a]P. In addition, several metabolic and signaling pathways were analyzed, these were used to compare the results of the toxic effect of AKRs on 1-NP and B[a]P. Through the application of kinase inhibitors, results indicated that p38-MAPK, but not ERK1/2 or JNK, was essential for mediating both 1-NP's and B[a]P's induction of the phosphorylation of p53 and cleavage of PARP. Neither ellipticine, a CYP1A1 inhibitor, nor 2,6-diisopropylphenol, a CYP1A2 and 2B1 inhibitor, blocked the toxic effects of 1-NP and B[a]P, which indicates that neither CYP1A1, 1A2, nor 2B1 is essential for the transformation of 1-NP and B[a], into toxic metabolites. AKR1C2 was constitutively expressed in HepG2 cells and was not regulated by 1-NP or B[a]P. In conclusion, this is the first report on AKRs' actions toward nitro-PAH in cells. The metabolic and signaling pathways for the toxic effects of both 1-NP and B[a]P are similar except that AKR1C2 plays differential role between them. The results provide valuable information for further investigations on AKRs.