SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8–43) and a short cytoplasmic helix (residues 53–60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6–18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5’ position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

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Over the last decade, the treatment paradigm of metastatic renal cell carcinoma has rapidly evolved, with notable changes in the front-line setting. Combination therapies involving the use of either doublet therapy with immune checkpoint inhibitors or combination VEGFR-directed therapies with immune checkpoint inhibitors have significantly improved clinical outcomes, including prolonged overall survival and durable response to treatment. We aim to highlight the Food and Drug Administration-approved front-line therapy options, the navigation of treatment selection, and the future directions of metastatic renal cell carcinoma therapies.

678 Background: Recent studies have suggested an association between the time-of-day of ICI infusions and disease outcomes, including progression free survival and overall survival, among patients with cancer. (Qian et al Lancet Oncology 2021). We sought to identify whether such an association exists in patients with mRCC receiving ICIs. Methods: Patients with mRCC treated with nivolumab alone, or in combination with ipilimumab, in either first- or second-line treatment were retrospectively identified. Patients who received <25% of infusions after 4:30 pm were assigned to the early time of infusion (TOI) sub-cohort while patients who received ≥ 25% of infusions after 4:30 pm were assigned to the late TOI sub-cohort. Objective response rate (ORR, per RECIST 1.1), time to treatment failure (TTF, defined as time from the date of first ICI infusion to time of treatment discontinuation), and overall survival (OS) were compared across the two groups using Cox proportional hazard models before and after adjustment for potential confounders (age, gender, line of treatment, IMDC risk, and histologic subtype). Results: A total of 145 mRCC pts (M:F,102:43) were included in the analysis. Median age was 64 (range 31-89) years, 81.4% had clear cell histology, and 75.9% had intermediate/poor risk disease. Early TOI sub-cohort included 110 (75.9%) patients while late TOI sub-cohort included 35 (24.1%) patients. Baseline characteristics were comparable across the two groups. Median OS for the entire cohort was 41.7 months (95% CI, 33.0 – Not reached [NR]), with a median TTF of 6.5 months (95% CI, 5.0 – 10.8). ORR was 32.7% in early TOI sub-group versus 25% in late TOI sub-group (p=0.60). Median TTF for the early TOI sub-cohort was 8.3 months (95% CI 6.0 – 12.6), as compared to 4.4 months (95% CI 2.1 - 10.8) among the late TOI group with a hazard ratio (HR) of 0.79 (95% CI, 0.50 – 1.25; p=0.32). Multivariate analysis showed a HR of 1.55 (95% CI, 0.98 – 2.57; p=0.06) after adjustment for potential confounders. The median OS was 46.3 months (95% CI, 32.2 – NR) in early TOI sub-cohort versus 41.7 months (95% CI, 16.7 – NR) in late TOI sub-group with a HR of 0.67 (95% CI 0.37 – 1.23; p=0.20) in univariate analyses and 0.61 (95% CI 0.33 – 1.15; p=0.13) in multivariate analyses. Conclusions: Our results demonstrated a numerical increase in ORR, TTF and OS with early TOI compared to late TOI, with the TTF difference approaching significance after adjustment for potential confounders. Larger randomized and controlled investigations are warranted to examine the impact of chronomodulation on the efficacy of ICIs in cancers, including mRCC.