SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

Small-molecule stabilization of protein-protein interactions is an emerging field in chemical biology. We show how fusicoccanes, originally identified as fungal toxins acting on plants, promote the interaction of 14-3-3 proteins with the human potassium channel TASK-3 and present a semisynthetic fusicoccane derivative (FC-THF) that targets the 14-3-3 recognition motif (mode 3) in TASK-3. In the presence of FC-THF, the binding of 14-3-3 proteins to TASK-3 was increased 19-fold and protein crystallography provided the atomic details of the effects of FC-THF on this interaction. We also tested the functional effects of FC-THF on TASK channels heterologously expressed in Xenopus oocytes. Incubation with 10 μM FC-THF was found to promote the transport of TASK channels to the cell membrane, leading to a significantly higher density of channels at the surface membrane and increased potassium current.

Cardiovascular disease is a major cause of death in chronic kidney disease (CKD) 2 (1). Atherosclerosis is highly prevalent in patients with severe renal failure and advances more rapidly in individuals with renal dysfunction compared with the general population (2). Reduced kidney function is associated with the risk of cardiovascular events, even when the dysfunction is mild (3).Leukocyte-endothelial interactions play an important role in the development of atherosclerosis (4). Cell adhesion molecules belonging to the immunoglobulin superfamily, such as ICAM-1 (intercellular cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1), together with members of the selectin family, including E-selectin, are upregulated to mediate monocyte/macrophage infiltration into atherosclerotic lesions (4, 5).Indoxyl sulfate is a uremic toxin synthesized in the liver from indole, a metabolite of tryptophan produced by the intestinal flora (6). In CKD patients, the serum levels of indoxyl sulfate are increased significantly compared with those in healthy individuals (7), and a number of studies have indicated that indoxyl sulfate accelerates glomerular sclerosis, whereas its accumulation promotes renal failure (8 -10). Other studies also showed that indoxyl sulfate induces endothelial dysfunction by releasing endothelial microparticles (11) and producing reactive oxygen species (ROS) (12). However, its effect on endothelial inflammatory processes such as leukocyte recruitment to vascular endothelium has not been reported.We report for the first time that indoxyl sulfate enhances monocyte adhesion to vascular endothelium through up-regulation of E-selectin and augmentation of oxidative stress in both in vitro and in vivo models. The underlying mechanisms seem to involve activation of JNK and NF-B. Our findings reveal a previously unrecognized molecular link between uremic toxins and cardiovascular diseases. EXPERIMENTAL PROCEDURESReagents-Indoxyl sulfate, N-acetylcysteine, probenecid, RPMI 1640 medium, and Dulbecco's PBS were obtained from Sigma. The JNK phosphorylation inhibitor SP600125, the p38 MAPK phosphorylation inhibitor SB203580, the ERK1/2 inhibitor U0126, and the IB phosphorylation inhibitor BAY11-7082 were purchased from Calbiochem. Recombinant human TNF-␣ was obtained from R&D Systems (Minneapolis, MN). A monoclonal antibody against E-selectin (clone 7A9) was obtained from American Type Culture Collection (Manassas, VA) (13). Antibodies against ICAM-1, VCAM-1, the NF-B p65 subunit, and the phospho-NF-B p65 subunit and a monoclonal blocking antibody against mouse E-selectin (clone UZ4) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK, anti-phospho-ERK, anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, and anti-phos-* This work was supported in part by Ministry of Education, Science, and Technology Grant-in-aid for Scientific Research 10178102 and special coordination funds, a grant-in-aid from the Ministry of Culture of Japan, a grant from the Ministry of Health, Labor, and We...

A cooperativity framework to describe and interpret small-molecule stabilization of protein–protein interactions (PPI) is presented, which allows elucidating structure–activity relationships regarding cooperativity and intrinsic affinity.