Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro.

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like protease (3CLpro) can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.50 Å resolution crystal structure of 3CLpro C145S bound to NEMO226-234 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for, in the pathology of COVID-19.

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro.

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 Å resolution crystal structure of 3CLpro C145S bound to NEMO 226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro- NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.

Potential Pathogenicity Determinants Identified from Structural Proteomics of SARS-CoV and SARS-CoV-2.

Despite SARS-CoV and SARS-CoV-2 being equipped with highly similar protein arsenals, the corresponding zoonoses have spread among humans at extremely different rates. The specific characteristics of these viruses that led to such distinct outcomes remain unclear. Here, we apply proteome-wide comparative structural analysis aiming to identify the unique molecular elements in the SARS-CoV-2 proteome that may explain the differing consequences. By combining protein modeling and molecular dynamics simulations, we suggest nonconservative substitutions in functional regions of the spike glycoprotein (S), nsp1, and nsp3 that are contributing to differences in virulence. Particularly, we explain why the substitutions at the receptor-binding domain of S affect the structure-dynamics behavior in complexes with putative host receptors. Conservation of functional protein regions within the two taxa is also noteworthy. We suggest that the highly conserved main protease, nsp5, of SARS-CoV and SARS-CoV-2 is part of their mechanism of circumventing the host interferon antiviral response. Overall, most substitutions occur on the protein surfaces and may be modulating their antigenic properties and interactions with other macromolecules. Our results imply that the striking difference in the pervasiveness of SARS-CoV-2 and SARS-CoV among humans seems to significantly derive from molecular features that modulate the efficiency of viral particles in entering the host cells and blocking the host immune response.

Potentially adaptive SARS-CoV-2 mutations discovered with novel spatiotemporal and explainable AI models.

BACKGROUND: A mechanistic understanding of the spread of SARS-CoV-2 and diligent tracking of ongoing mutagenesis are of key importance to plan robust strategies for confining its transmission. Large numbers of available sequences and their dates of transmission provide an unprecedented opportunity to analyze evolutionary adaptation in novel ways. Addition of high-resolution structural information can reveal the functional basis of these processes at the molecular level. Integrated systems biology-directed analyses of these data layers afford valuable insights to build a global understanding of the COVID-19 pandemic. RESULTS: Here we identify globally distributed haplotypes from 15,789 SARS-CoV-2 genomes and model their success based on their duration, dispersal, and frequency in the host population. Our models identify mutations that are likely compensatory adaptive changes that allowed for rapid expansion of the virus. Functional predictions from structural analyses indicate that, contrary to previous reports, the Asp614Gly mutation in the spike glycoprotein (S) likely reduced transmission and the subsequent Pro323Leu mutation in the RNA-dependent RNA polymerase led to the precipitous spread of the virus. Our model also suggests that two mutations in the nsp13 helicase allowed for the adaptation of the virus to the Pacific Northwest of the USA. Finally, our explainable artificial intelligence algorithm identified a mutational hotspot in the sequence of S that also displays a signature of positive selection and may have implications for tissue or cell-specific expression of the virus. CONCLUSIONS: These results provide valuable insights for the development of drugs and surveillance strategies to combat the current and future pandemics.

A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm.

Neither the disease mechanism nor treatments for COVID-19 are currently known. Here, we present a novel molecular mechanism for COVID-19 that provides therapeutic intervention points that can be addressed with existing FDA-approved pharmaceuticals. The entry point for the virus is ACE2, which is a component of the counteracting hypotensive axis of RAS. Bradykinin is a potent part of the vasopressor system that induces hypotension and vasodilation and is degraded by ACE and enhanced by the angiotensin1-9 produced by ACE2. Here, we perform a new analysis on gene expression data from cells in bronchoalveolar lavage fluid (BALF) from COVID-19 patients that were used to sequence the virus. Comparison with BALF from controls identifies a critical imbalance in RAS represented by decreased expression of ACE in combination with increases in ACE2, renin, angiotensin, key RAS receptors, kinogen and many kallikrein enzymes that activate it, and both bradykinin receptors. This very atypical pattern of the RAS is predicted to elevate bradykinin levels in multiple tissues and systems that will likely cause increases in vascular dilation, vascular permeability and hypotension. These bradykinin-driven outcomes explain many of the symptoms being observed in COVID-19.

In late 2019, a new virus named SARS-CoV-2, which causes a disease in humans called COVID-19, emerged in China and quickly spread around the world. Many individuals infected with the virus develop only mild, symptoms including a cough, high temperature and loss of sense of smell; while others may develop no symptoms at all. However, some individuals develop much more severe, life-threatening symptoms affecting the lungs and other parts of the body including the heart and brain. SARS-CoV-2 uses a human enzyme called ACE2 like a 'Trojan Horse' to sneak into the cells of its host. ACE2 lowers blood pressure in the human body and works against another enzyme known as ACE (which has the opposite effect). Therefore, the body has to balance the levels of ACE and ACE2 to maintain a normal blood pressure. It remains unclear whether SARS-CoV-2 affects how ACE2 and ACE work. When COVID-19 first emerged, a team of researchers in China studied fluid and cells collected from the lungs of patients to help them identify the SARS-CoV-2 virus. Here, Garvin et al. analyzed the data collected in the previous work to investigate whether changes in how the body regulates blood pressure may contribute to the life-threatening symptoms of COVID-19. The analyses found that SARS-CoV-2 caused the levels of ACE in the lung cells to decrease, while the levels of ACE2 increased. This in turn increased the levels of a molecule known as bradykinin in the cells (referred to as a 'Bradykinin Storm'). . Previous studies have shown that bradykinin induces pain and causes blood vessels to expand and become leaky which will lead to swelling and inflammation of the surrounding tissue. In addition, the analyses found that production of a substance called hyaluronic acid was increased and the enzymes that could degrade it greatly decreased. Hyaluronic acid can absorb more than 1,000 times its own weight in water to form a hydrogel. The Bradykinin-Storm-induced leakage of fluid into the lungs combined with the excess hyaluronic acid would likely result in a Jello-like substance that is preventing oxygen uptake and carbon dioxide release in the lungs of severely affected COVID-19 patients. Therefore, the findings of Garvin et al. suggest that the Bradykinin Storm may be responsible for the more severe symptoms of COVID-19. Further experiments identified several existing medicinal drugs that have the potential to be re-purposed to treat the Bradykinin Storm. A possible next step would be to carry out clinical trials to assess how effective these drugs are in treating patients with COVID-19. In addition, understanding how SARS-Cov-2 affects the body will help researchers and clinicians identify individuals who are most at risk of developing life-threatening symptoms.