Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed

Losartan inhibits sars-cov-2 replication in vitro

Purpose: SARS-CoV-2 infection is associated with substantial mortality and high morbidity. This study tested the effect of angiotensin II type I receptor blocker, losartan, on SARS-CoV-2 replication and inhibition of the papain-like protease of the virus. Methods: The dose-dependent inhibitory effect of losartan, in concentrations from 1μM to 100μM as determined by quantitative cell analysis combining fluorescence microscopy, image processing, and cellular measurements (Cellomics analysis) on SARS-CoV-2 replication was investigated in Vero E6 cells. The impact of losartan on deubiquitination and deISGylation of SARS-CoV-2 papain-like protease (PLpro) were also evaluated.  Results: Losartan reduced PLpro cleavage of tetraUbiquitin to diUbiquitin.  It was less effective in inhibiting PLpro’s cleavage of ISG15-AMC than Ubiquitin-AMC.  To determine if losartan inhibited SARS-CoV-2 replication, losartan treatment of SARS-CoV-2 infected Vero E6 was examined. Losartan treatment one hour prior to SARS-CoV-2 infection reduced levels of SARS-CoV-2 nuclear protein, an indicator of virus replication, by 80% and treatment one-hour post-infection decreased viral replication by 70%. Conclusion: Losartan was not an effective inhibitor of deubiquitinase or deISGylase activity of the PLpro but affected the SARS-CoV-2 replication of Vero E6 cells in vitro.  As losartan has a favorable safety profile and is currently available it has features necessary for efficacious drug repurposing and treatment of COVID-19

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6–8 amino acids. Short stretches of homologous host–pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations†

Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains

The vOTU Domain of Highly-pathogenic Porcine Reproductive and Respiratory Syndrome Virus Displays a Differential Substrate Preference

Arterivirus genus member Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically devastating disease, recently exacerbated by the emergence of highly pathogenic strains (HP-PRRSV). Within the nonstructural protein 2 of PRRSV is a deubiquitinating enzyme domain belonging to the viral ovarian tumor (vOTU) protease superfamily. vOTUs, which can greatly vary in their preference for their host ubiquitin (Ub) and Ub-like substrates such as interferon stimulated gene 15 (ISG15), have been implicated as a potential virulence factor. Since various strains of PRRSV have large variations in virulence, the specificity of vOTUs from two PRRSV strains of varying virulence were determined. While both vOTUs showed de-ubiquitinating activity and markedly low deISGylating activity, HP-PRRSV demonstrated a strong preference for lysine 63-linked poly-Ubiquitin, tied to innate immune response regulation. This represents the first report of biochemical activity unique to HP-PRRSV that has implications for a potential increase in immunosuppression and virulence

Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX

The enzyme organophosphorus acid anhydrolase (OPAA), from <i>Alteromonas</i> sp. JD6.5, has been shown to rapidly catalyze the hydrolysis of a number of toxic organophosphorus compounds, including several G-type chemical nerve agents. The enzyme was cloned into <i>Escherichia coli</i> and can be produced up to approximately 50% of cellular protein. There have been no previous reports of OPAA activity on VR {Russian VX, <i>O</i>-isobutyl <i>S</i>-[2-(diethylamino)­ethyl] methylphosphonothioate}, and our studies reported here show that wild-type OPAA has poor catalytic efficacy toward VR. However, via application of a structurally aided protein engineering approach, significant improvements in catalytic efficiency were realized via optimization of the small pocket within the OPAA’s substrate-binding site. This optimization involved alterations at only three amino acid sites resulting in a 30-fold increase in catalytic efficiency toward racemic VR, with a strong stereospecificity toward the P­(+) enantiomer. X-ray structures of this mutant as well as one of its predecessors provide potential structural rationales for their effect on the OPAA active site. Additionally, a fourth mutation at a site near the small pocket was found to relax the stereospecificity of the OPAA enzyme. Thus, it allows the altered enzyme to effectively process both VR enantiomers and should be a useful genetic background in which to seek further improvements in OPAA VR activity

A oncompetitive inhibitor for mycobacterium tuberculosis \u27s class iia fructose 1,6-bisphosphate Aldolase

Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 μM for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates. © 2013 American Chemical Society

Structural and Functional Characterization of Methicillin-Resistant Staphylococcus aureus’s Class IIb Fructose 1,6-Bisphosphate Aldolase

[Image: see text] Staphylococcus aureus is one of the most common nosocomial sources of soft-tissue and skin infections and has more recently become prevalent in the community setting as well. Since the use of penicillins to combat S. aureus infections in the 1940s, the bacterium has been notorious for developing resistances to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). With the persistence of MRSA as well as many other drug resistant bacteria and parasites, there is a growing need to focus on new pharmacological targets. Recently, class II fructose 1,6-bisphosphate aldolases (FBAs) have garnered attention to fill this role. Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA). With the recent finding of a flexible active site zinc-binding loop (Z-Loop) in class IIa FBAs and its potential for broad spectrum class II FBA inhibition, the lack of information regarding this feature of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop inhibitor development. Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA. Furthermore, we determined the K(M) for one of SaFBA’s substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop’s flexibility. Together the data offers insight into a class IIb FBA from a pervasively drug resistant bacterium and a comparison of Z-loops and other features between the different subtypes of class II FBAs

Development and validation of a yeast high-throughput screen for inhibitors of Aβ42 oligomerization

SUMMARY Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid β (Aβ), as the primary toxic species in Alzheimer’s disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aβ42 oligomer formation. Specifically, Aβ42 fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aβ42 oligomerization