An Evaluation of the Occupational Health Hazards of Peptide Couplers.

Peptide couplers (also known as amide bond-forming reagents or coupling reagents) are broadly used in organic chemical syntheses, especially in the pharmaceutical industry. Yet, occupational health hazards associated with this chemical class are largely unexplored, which is disconcerting given the intrinsic reactivity of these compounds. Several case studies involving occupational exposures reported adverse respiratory and dermal health effects, providing initial evidence of chemical sensitization. To address the paucity of toxicological data, a pharmaceutical cross-industry task force was formed to evaluate and assess the potential of these compounds to cause eye and dermal irritation as well as corrosivity and dermal sensitization. The goal of our work was to inform health and safety professionals as well as pharmaceutical and organic chemists of the occupational health hazards associated with this chemical class. To that end, 25 of the most commonly used peptide couplers and five hydrolysis products were selected for in vivo, in vitro, and in silico testing. Our findings confirmed that dermal sensitization is a concern for this chemical class with 21/25 peptide couplers testing positive for dermal sensitization and 15 of these being strong/extreme sensitizers. We also found that dermal corrosion and irritation (8/25) as well as eye irritation (9/25) were health hazards associated with peptide couplers and their hydrolysis products (4/5 were dermal irritants or corrosive and 4/5 were eye irritants). Resulting outcomes were synthesized to inform decision making in peptide coupler selection and enable data-driven hazard communication to workers. The latter includes harmonized hazard classifications, appropriate handling recommendations, and accurate safety data sheets, which support the industrial hygiene hierarchy of control strategies and risk assessment. Our study demonstrates the merits of an integrated, in vivo -in silico analysis, applied here to the skin sensitization endpoint using the Computer-Aided Discovery and REdesign (CADRE) and Derek Nexus programs. We show that experimental data can improve predictive models by filling existing data gaps while, concurrently, providing computational insights into key initiating events and elucidating the chemical structural features contributing to adverse health effects. This interactive, interdisciplinary approach is consistent with Green Chemistry principles that seek to improve the selection and design of less hazardous reagents in industrial processes and applications.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

Comparative evaluation of 11 in silico models for the prediction of small molecule mutagenicity: role of steric hindrance and electron-withdrawing groups.

The goal of this investigation was to perform a comparative analysis on how accurately 11 routinely-used in silico programs correctly predicted the mutagenicity of test compounds that contained either bulky or electron-withdrawing substituents. To our knowledge this is the first study of its kind in the literature. Such substituents are common in many pharmaceutical agents so there is a significant need for reliable in silico programs to predict precisely whether they truly pose a risk for mutagenicity. The predictions from each program were compared to experimental data derived from the Ames II test, a rapid reverse mutagenicity assay with a high degree of agreement with the traditional Ames assay. Eleven in silico programs were evaluated and compared: Derek for Windows, Derek Nexus, Leadscope Model Applier (LSMA), LSMA featuring the in vitro microbial Escherichia coli-Salmonella typhimurium TA102 A-T Suite (LSMA+), TOPKAT, CAESAR, TEST, ChemSilico (±S9 suites), MC4PC and a novel DNA docking model. The presence of bulky or electron-withdrawing functional groups in the vicinity of a mutagenic toxicophore in the test compounds clearly affected the ability of each in silico model to predict non-mutagenicity correctly. This was because of an over reliance on the part of the programs to provide mutagenicity alerts when a particular toxicophore is present irrespective of the structural environment surrounding the toxicophore. From this investigation it can be concluded that these models provide a high degree of specificity (ranging from 71% to 100%) and are generally conservative in their predictions in terms of sensitivity (ranging from 5% t o 78%). These values are in general agreement with most other comparative studies in the literature. Interestingly, the DNA docking model was the most sensitive model evaluated, suggesting a potentially useful new mode of screening for mutagens. Another important finding was that the combination of a quantitative structure-activity relationship and an expert rules system appeared to offer little advantage in terms of sensitivity, despite of the requirement for such a screening paradigm under the ICH M7 regulatory guideline.

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3ß.

BACKGROUND: New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities. METHODOLOGY/PRINCIPAL FINDINGS: In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3ß protein kinase isolated from T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3ß inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 µM). CONCLUSIONS/SIGNIFICANCE: Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3ß inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3ß inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.

Oculomotor deficits caused by 0.06% and 0.10% blood alcohol concentrations and relationship to subjective perception of drunkenness.

OBJECTIVE: The visual system is vital during critical activities such as driving. Studying how alcohol compromises the visual system physiologically is therefore important for safety reasons. The objective of the study was to investigate alcohol-related impairments in visual tasks performed under controlled breath alcohol concentrations (BAC) to determine dose-dependent effects. METHODS: Alcohol's effects on smooth pursuit and saccadic eye movements at 0.06% and 0.10% BAC were examined whilst recording alcohol levels by real-time measurements using a high precision breath analyzer. Oculomotor performance was recorded from 25 subjects by electronystagmography comprising measurements of smooth pursuit gain, saccade velocity, saccade accuracy and two novel parameters further describing oculomotor performance. RESULTS: Alcohol deteriorated accuracy of smooth pursuit movements (p<0.001) and saccadic velocities (p<0.01) at 0.06% BAC. At 0.10% BAC, smooth pursuit gains (p<0.01), saccade accuracies and saccade latencies (p<0.01) were also affected. The ratio between saccade velocity and saccade amplitude decreased significantly under alcohol intoxication (p<0.01). Self-perceptions of drunkenness correlated well with changes in smooth pursuit accuracy, but poorly with other oculomotor measures. CONCLUSIONS: Several of the smooth pursuit and saccade functions were altered dose-dependently by alcohol and small changes in BAC substantially changed the effects observed. Additionally, alcohol altered the relationship between saccade velocity and saccade amplitude, diminishing the capacity for saccades to reach high peak velocities. SIGNIFICANCE: The alcohol-induced oculomotor deficits, which were found already at 0.06% BAC by our more sensitive analysis methods, may have safety implications for tasks that rely on visual motor control and visual feedback.

On developing a process for conducting extractable-leachable assessment of components used for storage of biopharmaceuticals.

Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts. Adducts and leachables may or may not be detected as product-related impurities in routine stability indicating assays depending on the rigor of the analytical program. The need for the development of a thorough and holistic extractable and leachable program based on risk assessment, review of existing literature, and consolidation of industry best practices is discussed. Standardizing component use within an organization enables streamlining of the extractable-leachable program. Our strategy for an extractable-leachable program is divided into different stages, each stage detailing the activities and the department within the organization that is responsible for execution of these activities. The roles and responsibilities of the key stakeholders are identified. The integration of analytical activities with health-based risk-assessment information into the design of an extractable-leachable program is highlighted.

Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics.

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range. Assessment of isotope ratio measurement variability suggests substantial contributions from natural measurement variability. A better understanding of this variable will allow assessment of the contribution of sequence dependence. Both variables must be understood before meaningful mixing experiments for relative expression proteomics are performed. Subtle modification of isotope ratio ( approximately 1-2% increase in (13)C) had no effect upon either the ability of data-dependent acquisition software or database searching software to trigger tandem mass spectrometry or match MSMS data to peptide sequences. More severe modification of isotope ratio caused a significant drop in performance of both functionalities. Development of software for deconvolution of isotope ratio concomitant with protein identification using LC-MSMS, or any other proteomics strategy, is underway (Isosolv). The identified peptide sequence is then be used to provide elemental composition for accurate isotope ratio decoding and the potential to control for specific amino acid biases should these prove significant. It is suggested that subtle modification of isotope ratio proteomics (SMIRP) offers a convenient approach to in vivo isotope coding of plants and might ultimately be extended to mammals including humans.

Transit peptide cleavage sites of integral thylakoid membrane proteins.

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.

The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry.

Proteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications. Provisional identity of 30 of these gene products was proposed based upon coincidence of measured mass with that calculated from genomic sequence. Analysis of isolated photosystem II complexes allowed detection and resolution of a minor population of D1 (PsbA) that was apparently palmitoylated and not detected in less purified preparations. Based upon observed +80-Da adducts, D1, D2 (PsbD), CP43 (PsbC), two Lhcbs, and PsbH were confirmed to be phosphorylated, and a new phosphoprotein was proposed to be the product of psbT. The appearance of a second +80-Da adduct on PsbH provides direct evidence for a second phosphorylation site on PsbH, complicating interpretation of its role in regulation of thylakoid membrane organization and function, including light-state transitions. Adducts of +32 Da, presumably arising from oxidative modification during illumination, were associated with more highly phosphorylated forms of PsbH implying a relationship between the two phenomena. Intact mass proteomics of organellar subfractions and more highly purified protein complexes provides increasingly detailed insights into functional genomics of photosynthetic membranes.