Machine Learning Guided Design of High-Affinity ACE2 Decoys for SARS-CoV-2 Neutralization.

A potential therapeutic strategy for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding to the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant variant, named sACE22.v.2.4, that exhibits subnanomolar affinity to the receptor-binding domain (RBD) of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identify other ACE2 variants that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified multiple ACE2 double mutants that bind SARS-CoV-2 S with tighter affinity as compared to the wild type, most notably L79V;N90D that binds RBD similarly to ACE22.v.2.4. The experimental validation of the double mutants successfully demonstrates the use of machine learning approaches for engineering protein-protein interactions and identifying high-affinity ACE2 peptides for targeting SARS-CoV-2.

Control of SARS-CoV-2 infection by MT1-MMP-mediated shedding of ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for SARS-CoV-2. The full-length membrane form of ACE2 (memACE2) undergoes ectodomain shedding to generate a shed soluble form (solACE2) that mediates SARS-CoV-2 entry via receptor-mediated endocytosis. Currently, it is not known how the physiological regulation of ACE2 shedding contributes to the etiology of COVID-19 in vivo. The present study identifies Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) as a critical host protease for solACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 infection leads to increased activation of MT1-MMP that is colocalized with ACE2 in human lung epithelium. Mechanistically, MT1-MMP directly cleaves memACE2 at M706-S to release solACE218-706 that binds to the SARS-CoV-2 spike proteins (S), thus facilitating cell entry of SARS-CoV-2. Human solACE218-706 enables SARS-CoV-2 infection in both non-permissive cells and naturally insusceptible C57BL/6 mice. Inhibition of MT1-MMP activities suppresses solACE2-directed entry of SARS-CoV-2 in human organoids and aged mice. Both solACE2 and circulating MT1-MMP are positively correlated in plasma of aged mice and humans. Our findings provide in vivo evidence demonstrating the contribution of ACE2 shedding to the etiology of COVID-19.

Targeting epigenetic regulators for inflammation: Mechanisms and intervention therapy.

Emerging evidence indicates that resolution of inflammation is a critical and dynamic endogenous process for host tissues defending against external invasive pathogens or internal tissue injury. It has long been known that autoimmune diseases and chronic inflammatory disorders are characterized by dysregulated immune responses, leading to excessive and uncontrol tissue inflammation. The dysregulation of epigenetic alterations including DNA methylation, posttranslational modifications to histone proteins, and noncoding RNA expression has been implicated in a host of inflammatory disorders and the immune system. The inflammatory response is considered as a critical trigger of epigenetic alterations that in turn intercede inflammatory actions. Thus, understanding the molecular mechanism that dictates the outcome of targeting epigenetic regulators for inflammatory disease is required for inflammation resolution. In this article, we elucidate the critical role of the nuclear factor-κB signaling pathway, JAK/STAT signaling pathway, and the NLRP3 inflammasome in chronic inflammatory diseases. And we formulate the relationship between inflammation, coronavirus disease 2019, and human cancers. Additionally, we review the mechanism of epigenetic modifications involved in inflammation and innate immune cells. All that matters is that we propose and discuss the rejuvenation potential of interventions that target epigenetic regulators and regulatory mechanisms for chronic inflammation-associated diseases to improve therapeutic outcomes.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Effects of Chinese medicine for COVID-19 rehabilitation: a multicenter observational study.

OBJECTIVES: This study aimed to evaluate the effects of Chinese Medicine (CM) on the health condition of the post-COVID-19 patients, particularly with the CM Syndrome diagnosis and Body Constitutions (BC), as well as related clinical characteristics. METHODS: 150 participants who had COVID-19 and discharged from Hong Kong public hospitals were recruited. They were provided with three to six months of CM treatments, during which assessments were made per month and at follow-up on their CM syndromes, BC, lung functions, and other medical conditions. This study was divided into two parts: (1) Retrospective survey: medical history of participants during COVID-19 hospitalization was collected during the baseline visit; (2) Prospective observation and assessments: clinical symptoms, lung functions, and BC status were evaluated in participants receiving CM treatment based on syndrome differentiation and clinical symptoms. RESULTS: The median hospitalization period was 16 days. Symptoms were presented in 145 (96.6%) patients at the day they were diagnosed with COVID-19. Fever, fatigue, and dry cough were the most common symptoms, exhibiting in 59.3% (89 of 150), 55.3% (83 of 150), and 46% (70 of 150) participants, respectively. Among the 150 post-COVID patients, majority (71.3%) were of the two particular post-COVID CM Syndromes (Qi Deficiency of Lung and Spleen, and Qi and Yin Deficiency). Upon CM treatment, there was an observable increase in participants reaching a balanced BC (i.e. healthy body conditions). The increase was observed to be more prominent in those without the particular CM Syndromes compared to those with the CM Syndromes. Main clinical symptoms in participants with the CM Syndromes decreased upon CM treatment. Occurrence of fatigue also dropped after CM treatment though not all accompanied clinical symptoms were resolved fully. Further to the improvement in terms of CM assessments, lung functions of the participants were found to show improvement after treatment. Both the performance in 6MWT and scores in the LFQ improved upon CM treatments (P < 0.05). CONCLUSION: This study provided evidence for individualized CM treatment on COVID-19 rehabilitation concerning the clinical symptoms improvements, lung functions improvement, and achieving a balanced BC. It is believed that CM may be a key to further promote rehabilitation and resolution of residual symptoms. Long-term large scale follow-up studies on sub-categorising post-COVID patients according to different CM syndromes would be required to further elucidate treatment of persistent symptoms that may be associated with long-COVID.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Computationally Designed ACE2 Decoy Receptor Binds SARS-CoV-2 Spike (S) Protein with Tight Nanomolar Affinity.

Even with the availability of vaccines, therapeutic options for COVID-19 still remain highly desirable, especially in hospitalized patients with moderate or severe disease. Soluble ACE2 (sACE2) is a promising therapeutic candidate that neutralizes SARS CoV-2 infection by acting as a decoy. Using computational mutagenesis, we designed a number of sACE2 derivatives carrying three to four mutations. The top-predicted sACE2 decoy based on the in silico mutagenesis scan was subjected to molecular dynamics and free-energy calculations for further validation. After illuminating the mechanism of increased binding for our designed sACE2 derivative, the design was verified experimentally by flow cytometry and BLI-binding experiments. The computationally designed sACE2 decoy (ACE2-FFWF) bound the receptor-binding domain of SARS-CoV-2 tightly with low nanomolar affinity and ninefold affinity enhancement over the wild type. Furthermore, cell surface expression was slightly greater than wild-type ACE2, suggesting that the design is well-folded and stable. Having an arsenal of high-affinity sACE2 derivatives will help to buffer against the emergence of SARS CoV-2 variants. Here, we show that computational methods have become sufficiently accurate for the design of therapeutics for current and future viral pandemics.

An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.