Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase

Thimet oligopeptidase is a metalloenzyme involved in regulatingneuropeptide processing. Three cysteine residues (246, 248,253) are known to be involved in thiol activation of the enzyme.In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S)displays increased activity in the absence of dithiothreitol.Dimers, purportedly formed through cysteines 246, 248 and 253,have been thought to be inactive. However, analysis of the triplemutant by native gel electrophoresis reveals the existence ofdimers and multimers, implying that oligomer formation is mediatedby other cysteines, probably on the surface, and that some ofthese forms are enzymatically active. Isolation and characterizationof iodoacetate-modified monomers and dimers of the triple mutantrevealed that, indeed, certain dimeric forms of the enzyme arestill fully active, whereas others show reduced activity. Cysteineresidues potentially involved in dimerization were identifiedby modeling of thimet oliogopeptidase to its homolog, neurolysin.Five mutants were constructed; all contained the triple mutationC246S/C248S/C253S and additional substitutions. Substitutionsat C46 or C682 and C687 prevented multimer formation and inhibiteddimer formation. The C46S mutant had enzymatic activity comparableto the parent triple mutant, whereas that of C682S/C687S wasreduced. Thus, the location of intermolecular disulfide bonds,rather than their existence per se, is relevant to activity.Dimerization close to the N-terminus is detrimental to activity,whereas dimerization near the C-terminus has little effect.Altering disulfide bond formation is a potential regulatoryfactor in the cell owing to the varying oxidation states insubcellular compartments and the different compartmental locationsand functions of the enzyme. Received March 1, 2003; revised June 17, 2003; accepted June 23, 2003.     

Flow influenced electrochemical corrosion of nickel aluminium bronze – Part I. Cathodic polarisation

The cathodic polarisation behaviour of CA 104 nickel aluminium bronze (NAB) has been examined in fully characterised seawaters (filtered and artificial) using the rotating disc electrode (RDE) and the rotating cylinder electrode (RCE). Linear sweep voltammetry and a potential step, current transient technique were used to examine the charge transfer and mass transfer controlled cathodic response as a function of both laminar and turbulent fluid flow. For freshly polished surfaces, the rate of irreversible charge transfer controlled oxygen reduction is controlled by the exchange of a single electron and hydrogen evolution is only significant at potentials more negative than approximately –1.0 V vs. the saturated calomel electrode (SCE).     

Non-heme Fe(IV)-oxo intermediates

High-valent non-heme iron-oxo intermediates have been proposed for decades as the key intermediates in numerous biological oxidation reactions. In the past three years, the first direct characterization of such intermediates has been provided by studies of several alphaKG-dependent oxygenases that catalyze either hydroxylation or halogenation of their substrates. In each case, the Fe(IV)-oxo intermediate is implicated in cleavage of the aliphatic C-H bond to initiate hydroxylation or halogenation. The observation of non-heme Fe(IV)-oxo intermediates and Fe(II)-containing product(s) complexes with almost identical spectroscopic parameters in the reactions of two distantly related alphaKG-dependent hydroxylases suggests that members of this subfamily follow a conserved mechanism for substrate hydroxylation. In contrast, for the alphaKG-dependent non-heme iron halogenase, CytC3, two distinct Fe(IV) complexes form and decay together, suggesting that they are in rapid equilibrium. The existence of two distinct conformers of the Fe site may be the key factor accounting for the divergence of the halogenase reaction from the more usual hydroxylation pathway after C-H bond cleavage. Distinct transformations catalyzed by other mononuclear non-heme enzymes are likely also to involve initial C-H bond cleavage by Fe(IV)-oxo complexes, followed by diverging reactivities of the resulting Fe(III)-hydroxo/substrate radical intermediates.     

Impact of Inspected Buffers on Production Parameters of Construction Processes

This paper examines the production implications of quality control inspections conducted on the buffer between processes in a construction project by modeling the linkage between these processes. Inspection of partially completed work at the end of one activity but before the beginning of work by the next activity is fairly common. Work that is deemed to be of sufficient quality is then made available for the next activity. Work that is deemed insufficient requires rework, typically by the trade appropriate to the activity that fed into the buffer, to bring the work into compliance. This has implications for workload management for that trade, of course, as well as for the reliability of work flow to the successor or downstream processes. While such situations are common in all construction sectors, an example from the residential construction sector was examined via a simulation model augmented by field data collected from residential construction projects. The impacts of the work flow into the predecessor process, the inspection pass rate, and resource availability were examined. The inspection pass rate was found to dramatically affect the reliability of work flow, unless resources are unlimited. Furthermore, the inspection pass rate was found to be functionally related to the production parameters of the process.     

The Analysis of Metal Ions in Solution

Established methods for the chemical analysis of dissolved metal are concisely reviewed. In each case, the principles of a given technique are considered and its relative merits are outlined.     

Moving out of the red zone: addressing staff allocation to improve patient satisfaction.

BACKGROUND: Each summer, Hermann Hospital (Houston), like virtually all health care organizations, faces staffing challenges because of employee vacations, increased patient load, and staff turnover. A "zone" system was developed to address staff allocation, which was identified as a factor in deterioration of the hospital's patient satisfaction performance. ZONE SYSTEM: Every day, each unit or department designated the zone most appropriate based on factors identified in root cause analysis--high patient census, high patient acuity, emergent activity, and the number of float, agency, or unfamiliar staff members. A green zone defines conditions where the staff is very comfortable; yellow reflects increased activity; and red indicates that staff members are stressed and overwhelmed with patient needs. ACTION STEPS: Management's action steps included decreasing the nursing vacancy rate to minimize reliance on agency and float staff members and securing longer-term commitments from temporary staff members. Individual units also generated contingency plans, such as identifying a "partner unit" to provide shared staff members, supplies, and other assistance. RESULTS: The percentage of patients rating their overall experience at Hermann Hospital as "good" or "excellent" increased from 83% in August 1997 to 89% in August 1998, despite increases in admissions, patient days, operating room cases, and emergency department visits. DISCUSSION: The zone system provides a rapid way to quantify contributing factors to patient dissatisfaction and respond to them. Hermann Hospital is currently developing a broader zone system to include staff vacancy rates by departments, areas of increased patient census for prolonged periods, and balancing episodic zones with prolonged zones.     

Power spectral density analysis of optical substrates for gravitational-wave interferometry

The power spectral density of surface-relief variations on polished optical surfaces across microscopic through to macroscopic spatial scales is calculated from measurements on substrates that are being produced for the Laser Interferometer Gravitational-Wave Observatory (LIGO). These spectra give a guide to the scattering properties of the surface, which in turn critically influence the performance of LIGO. Measurements obtained by use of a full-aperture interferometer and an interference microscope with two different objectives are combined to produce one-dimensional power spectral density representations of the surfaces across spatial frequencies ranging from 0.1 to 8000 cm(-1). These measurements from different instruments are in good agreement with an analytic power spectrum that varies as nu(-1.5), where nu is the spatial frequency. Some anomalies in the power spectral density spectra can be related to aspects of the polishing process.     

Polarization discrimination of coherently propagating light in turbid media

We describe the use of degree of polarization to discriminate unscattered and weakly scattered light from multiply scattered light in an optically turbid material. We use spatially resolved measurements of the degree of polarization to compare how well linearly and circularly polarized light survives in a sample. Experiments were performed on common tissue phantoms consisting of polystyrene and Intralipid microsphere suspensions and on adipose and arterial tissue. The results indicate that polarization is maintained even after unpolarized irradiance through each sample has been extinguished by several orders of magnitude. The results also show that polarized light propagation in common tissue phantoms is distinctly different from polarized light propagation in the two tissues investigated. Further, these experiments illustrate when polarization is an effective discrimination criterion and when it is not. The potential of a polarization-based discrimination scheme to image through the biological and nonbiological samples investigated here is also discussed.