Carly Huitema

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase.

Abstract The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5–7 μM) toward SARS-CoV 3CLpro.

Crystal Structures of the Main Peptidase from the SARS Coronavirus Inhibited by a Substrate-like Aza-peptide Epoxide

The main peptidase (Mpro) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive molecular targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV Mpro by an aza-peptide epoxide (APE; k inact/K i=1900(±400)M−1 s−1). The crystal structures of the Mpro:APE complex in the space groups C2 and P212121 revealed the formation of a covalent bond between the catalytic Cys145 Sγ atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of Mpro in a substrate-like manner, with excellent structural and chemical complementarity. In addition, the crystal structure of unbound Mpro in the space group C2 revealed that the “N-fingers” (N-terminal residues 1 to 7) of both protomers of Mpro are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystallization pH of 6.5, contrary to the previously determined crystal structures of unbound Mpro in the space group P21.

A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase

Abstract The 3C-like main peptidase 3CLpro is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CLpro and the structurally related 3Cpro viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CLpro in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzymes catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.

Assessment of activated sludge microbial community analysis in full-scale biological wastewater treatment plants using patterns of fatty acid isopropyl esters (FAPEs)

This investigation introduces the application of a relatively rapid technique to obtain information about the dynamic nature of microbial communities in activated sludge. The objective has been to consider variability due to measurement errors and protocol changes within the same quantitative framework as the analysis of systematic differences in microbial communities in large-scale aerobic activated sludge secondary wastewater treatment systems. Adjustments to the methodology were considered due to their potential for simplifying and shortening the analysis procedure. All modifications to the protocols used to assay the composition of microbial fatty acids (MFAs) of activated sludge imposed some bias to the chromatographic data. This methodological bias was similar in magnitude to the level of discrimination between activated sludge microbial community structures that were considered as part of the present study. MFA analysis supported the expectations of subtle but systematic community structure differences and shifts in activated sludge based on the current understanding of these wastewater treatment systems. A standardized MFA methodology was shown to be sensitive to minor systematic changes in activated sludge communities due the anticipated underlying factors of selective pressures from the process configuration, history, operational conditions and/or nutrient status. The chemometric approach of fatty acid isopropyl ester analysis of activated sludge can provide a routine tool for meaningful and quantitative information of changes in activated sludge quality in full-scale treatment systems.

Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase.

Abstract The severe acute respiratory syndrome (SARS) virus depends on a chymotrypsin-like cysteine proteinase (3CLpro) to process the translated polyproteins to functional viral proteins. This enzyme is a target for the design of potential anti-SARS drugs. A series of ketones and corresponding mono- and di-fluoro ketones having two or three aromatic rings were synthesized as possible reversible inhibitors of SARS 3CLpro. The design was based on previously established potent inhibition of the enzyme by oxa analogues (esters), which also act as substrates. Structure–activity relationships and modeling studies indicate that three aromatic rings, including a 5-bromopyridin-3-yl moiety, are key features for good inhibition of SARS 3CLpro. Compound 11d, 2-(5-bromopyridin-3-yl)-1-(5-(4-chlorophenyl)furan-2-yl)ethanone and its α-monofluorinated analogue 12d, gave the best reversible inhibition with IC50 values of 13μM and 28μM, respectively. In contrast to inhibitors having two aromatic rings, α-fluorination of compounds with three rings unexpectedly decreased the inhibitory activity.

Heteroaromatic ester inhibitors of hepatitis A virus 3C proteinase : Evaluation of mode of action

Abstract The related 3C and 3C-like proteinase (3Cpro and 3CLpro) of picornaviruses and coronaviruses, respectively, are good drug targets. As part of an effort to generate broad-spectrum inhibitors of these enzymes, we screened a library of inhibitors based on a halopyridinyl ester from a previous study of the severe acute respiratory syndrome (SARS) 3CL proteinase against Hepatitis A virus (HAV) 3Cpro. Three of the compounds, which also had furan rings, inhibited the cleavage activity of HAV 3Cpro with K ics of 120–240nM. HPLC-based assays revealed that the inhibitors were slowly hydrolyzed by both HAV 3Cpro and SARS 3CLpro, confirming the identity of the expected products. Mass spectrometric analyses indicated that this hydrolysis proceeded via an acyl-enzyme intermediate. Modeling studies indicated that the halopyridinyl moiety of the inhibitor fits tightly into the S1-binding pocket, consistent with the lack of tolerance of the inhibitors to modification in this portion of the molecule. These compounds are among the most potent non-peptidic inhibitors reported to date against a 3Cpro.

Simulated Microgravity (SMG) and Bacteria

This past century has been a scientific revolution in the understanding of the cell as the basic unit of life. However an immense paucity of knowledge exists on microbial growth, survival, function and structure in space. However, there are significant constraints placed on conducting biological research in space such as time, available stowage space, trained personnel, power requirements, weight and the possibility of accidental microbiological contamination. One Earth-based approach is to use a modification of a clinostat known as a HARV (high-aspect-ratio-vessel; Synthecon Inc., Houston, Texas, USA) to conduct this research. In this note we describe the use of the HARV to examine the effects of randomized microgravity (RMG) on bacterial growth and membrane polarization.

A Fluorescent Protein-Based Biological Screen of Proteinase Activity

A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3Cpro, prefers Ile, Val, or Leu at the P4 position of the cleavage sequence and Gly, Ser, or Ala at the P′1 position. The assay can also be used to screen small-molecule libraries for inhibitors.

Analyzing Enzyme Kinetic Data Using the Powerful Statistical Capabilities of R

We describe a powerful tool for enzymologists to use for typical non-linear fitting of common equations in enzyme kinetics using the statistical program R. Enzyme kinetics is a powerful tool for understanding enzyme catalysis, regulation, and inhibition but tools to perform the analysis have limitations. Software to perform the necessary nonlinear analysis may be proprietary, expensive or difficult to use, especially for a beginner. The statistical program R is ideally suited to analyzing enzyme kinetic data; it is free in two respects: there is no cost and there is freedom to distribute and modify. It is also robust, powerful and widely used in other fields of biology. In this paper we introduce the program R to enzymologists who want to analyze their data but are unfamiliar with R or similar command line statistical analysis programs. Data are inputted and examples of different non-linear models are fitted. Results are extracted and plots are generated to assist judging the goodness of fit. The instructions will allow users to create their own modifications to adapt the protocol to their own experiments. Because of the use of scripts, a method can be modified and used to analyze different datasets in less than one hour.