Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

Reactivity and mechanism in chemical and synthetic biology.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20-21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism. This article is part of the themed issue 'Reactivity and mechanism in chemical and synthetic biology'.

ALLOSTERY to DISRUPT PROTEIN-PROTEIN INTERACTIONS with SMALL MOLECULES

It is commonly considered that protein-protein interactions are extremely difficult to target using small molecules. Our group uses a chemical biology approach to study the allosteric, regulatory, mechanisms of protein kinases involved in growth factor signaling downstream of PI3-kinase. Over the years we have described small molecules that target a regulatory site in AGC kinases called "PIF-Pocket", and allosterically affect the active site, ATP-binding site. We also have shown that molecules that bind to the active site of protein kinases can stimulate or inhibit protein-protein interactions at the PIF-pocket regulatory site, by a mechanism that we have termed "reverse allostery". The reverse allosteric effects are widely produced by protein kinases inhibitors that target the ATP-binding site, although the phenomenon has been almost completely unperceived by pharmaceutical industries. The presence of a reverse allosteric effect is also the cause of the paradoxical effects on the protein kinases signal transduction detected for certain inhibitors. Thus, using the knowledge of the molecular mechanism involved in the bidirectional allostery, it becomes possible to exploit it to break protein-protein interactions between protein kinases in their protein complexes (Trends Biochem Sci 45(1):27-41, 2020). We will summarize our detailed research on the protein kinase PDK1 as a model of allosteric protein and will analyze the results in the context of the modern models of allostery, "conformational selection" and "population shift". We suggest that the principles of allostery should be used to rationalize new approaches to push forward the discovery of novel drugs that break proteinprotein interactions. In line with the allosteric mechanism to disrupt protein kinase interactions, we also hypothesized that such a mechanism could also be used to break the interactions between other proteins, for example, between the angiotensin converting enzyme II (ACE2) and SARS-CoV-2 spike protein, which could be used as treatment against coronavirus infection (ChemMedChem. 15(18):1682-1690, 2020). Current studies confirm that compounds with allosteric mechanism can indeed disrupt the interaction between ACE2 and Spike. Enzymology.

In Vitro Evaluation of In Silico Screening Approaches in Search for Selective ACE2 Binding Chemical Probes.

New models for ACE2 receptor binding, based on QSAR and docking algorithms were developed, using XRD structural data and ChEMBL 26 database hits as training sets. The selectivity of the potential ACE2-binding ligands towards Neprilysin (NEP) and ACE was evaluated. The Enamine screening collection (3.2 million compounds) was virtually screened according to the above models, in order to find possible ACE2-chemical probes, useful for the study of SARS-CoV2-induced neurological disorders. An enzymology inhibition assay for ACE2 was optimized, and the combined diversified set of predicted selective ACE2-binding molecules from QSAR modeling, docking, and ultrafast docking was screened in vitro. The in vitro hits included two novel chemotypes suitable for further optimization.

CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting.

BACKGROUND: The well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-PCR assays has led to the use of alternate testing options. Here we evaluate the accuracy and performance of a novel fluorescence-based assay that received EUA authorization for detecting SARS-CoV-2 in clinical samples. METHODS: The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-PCR assays. Positive samples were selected based on a broad range of cycle thresholds. RESULTS: A total of 60 COVID-19 patient samples were correctly diagnosed with 100% detection accuracy (relative fluorescence ratios: N gene 95% CI 29.9-43.8, ORF1ab gene 95% CI 30.1-46.3). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence. CONCLUSIONS: We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads, including low titers, using SHERLOCK CRISPR collateral detection in a clinical setting. These findings demonstrate encouraging results, at a time when COVID-19 clinical diagnosis and screening protocols remain in demand; especially as new variants emerge and vaccine mandates evolve. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.

Lactate dehydrogenase: an old enzyme reborn as a COVID-19 marker (and not only).

Background Historically, the lactate dehydrogenase (LDH) measurement was introduced into Laboratory Medicine as component (together with creatine kinase (CK) and aspartate aminotransferase) of the classical enzyme triad employed for the diagnosis of myocardial infarction, which was subsequently replaced by CK-MB, and more recently by cardiac troponins. Afterwards, for many years, the clinical application of serum LDH measurement has been limited to the evaluation of anemias and to as a rough prognostic tool for certain tumors. Content In the last few years, significant changes have happened. First, the test has been confirmed as a robust predictor of poor outcomes in many neoplastic conditions. Furthermore, in the Revised International Staging System adopted in the 2015 by the International Myeloma Working Group, LDH acts as determinant of disease biology in differentiating myeloma stages. Finally, in the last few months, LDH is definitively reborn given its proven significant contribution in defining the COVID-19 severity. Conclusions This increased clinical role calls for an improvement of LDH assay standardization through the implementation of traceability of results of clinical samples to the available reference measurement system.