Comprehensive chemical characterization of the aerosol generated by a heated tobacco product by untargeted screening.

A suite of untargeted methods has been applied for the characterization of aerosol from the Tobacco Heating System 2.2 (THS2.2), a heated tobacco product developed by Philip Morris Products S.A. and commercialized under the brand name IQOS®. A total of 529 chemical constituents, excluding water, glycerin, and nicotine, were present in the mainstream aerosol of THS2.2, generated by following the Health Canada intense smoking regimen, at concentrations ≥ 100 ng/item. The majority were present in the particulate phase (n = 402), representing more than 80% of the total mass determined by untargeted screening; a proportion were present in both particulate and gas-vapor phases (39 compounds). The identities for 80% of all chemical constituents (representing > 96% of the total determined mass) were confirmed by the use of authentic analytical reference materials. Despite the uncertainties that are recognized to be associated with aerosol-based untargeted approaches, the reported data remain indicative that the uncharacterized fraction of TPM generated by THS2.2 has been evaluated to the fullest practicable extent. To the best of our knowledge, this work represents the most comprehensive chemical characterization of a heated tobacco aerosol to date. Graphical abstract.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

Performance Evaluation of a Nontargeted Platform Using Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry Integrating Computer-Assisted Structure Identification and Automated Semiquantification for the Comprehensive Chemical Characterization of a Complex Matrix.

Nontargeted screening methodologies are powerful approaches for comprehensive chemical characterization of complex matrixes. In order to maximize chemical space coverage, three analytical methods using two-dimensional gas chromatography with time-of-flight mass spectrometry for nonpolar, polar, and volatile compounds have been established. The structural identification process was streamlined with an in-house developed computer-assisted structure identification platform, which facilitated the identification of novel compounds and also delivered semiquantitative concentrations for all compounds. Key performance parameters for this nontargeted platform, including chemical space coverage, confidence for structural identification, accuracy of semiquantification, and performance of differential analysis, were evaluated. The automated structural identification process was assessed using a subset of 243 compounds (out of 2990), which were confirmed to be present in cigarette smoke using reference standards. Consistently high true positive identification rates between 88.2% and 96.2% across the different concentration ranges investigated were demonstrated. Accuracy for semiquantification was assessed by comparison with quantitative data from literature, where a maximum 4-fold deviation from available targeted analysis values was estimated.

Prediction Models of Retention Indices for Increased Confidence in Structural Elucidation during Complex Matrix Analysis: Application to Gas Chromatography Coupled with High-Resolution Mass Spectrometry.

Monitoring of volatile and semivolatile compounds was performed using gas chromatography (GC) coupled to high-resolution electron ionization mass spectrometry, using both headspace and liquid injection modes. A total of 560 reference compounds, including 8 odd n-alkanes, were analyzed and experimental linear retention indices (LRI) were determined. These reference compounds were randomly split into training (n = 401) and test (n = 151) sets. LRI for all 552 reference compounds were also calculated based upon computational Quantitative Structure-Property Relationship (QSPR) models, using two independent approaches RapidMiner (coupled to Dragon) and ACD/ChromGenius software. Correlation coefficients for experimental versus predicted LRI values calculated for both training and test set compounds were calculated at 0.966 and 0.949 for RapidMiner and at 0.977 and 0.976 for ACD/ChromGenius, respectively. In addition, the cross-validation correlation was calculated at 0.96 from RapidMiner and the residual standard error value obtained from ACD/ChromGenius was 53.635. These models were then used to predict LRI values for several thousand compounds reported present in tobacco and tobacco-related fractions, plus a range of specific flavor compounds. It was demonstrated that using the mean of the LRI values predicted by RapidMiner and ACD/ChromGenius, in combination with accurate mass data, could enhance the confidence level for compound identification from the analysis of complex matrixes, particularly when the two predicted LRI values for a compound were in close agreement. Application of this LRI modeling approach to matrixes with unknown composition has already enabled the confirmation of 23 postulated compounds, demonstrating its ability to facilitate compound identification in an analytical workflow. The goal is to reduce the list of putative candidates to a reasonable relevant number that can be obtained and measured for confirmation.

Computer-assisted structure identification (CASI)--an automated platform for high-throughput identification of small molecules by two-dimensional gas chromatography coupled to mass spectrometry.

Compound identification is widely recognized as a major bottleneck for modern metabolomic approaches and high-throughput nontargeted characterization of complex matrices. To tackle this challenge, an automated platform entitled computer-assisted structure identification (CASI) was designed and developed in order to accelerate and standardize the identification of compound structures. In the first step of the process, CASI automatically searches mass spectral libraries for matches using a NIST MS Search algorithm, which proposes structural candidates for experimental spectra from two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) measurements, each with an associated match factor. Next, quantitative structure-property relationship (QSPR) models implemented in CASI predict three specific parameters to enhance the confidence for correct compound identification, which were Kovats Index (KI) for the first dimension (1D) separation, relative retention time for the second dimension separation (2DrelRT) and boiling point (BP). In order to reduce the impact of chromatographic variability on the second dimension retention time, a concept based upon hypothetical reference points from linear regressions of a deuterated n-alkanes reference system was introduced, providing a more stable relative retention time measurement. Predicted values for KI and 2DrelRT were calculated and matched with experimentally derived values. Boiling points derived from 1D separations were matched with predicted boiling points, calculated from the chemical structures of the candidates. As a last step, CASI combines the NIST MS Search match factors (NIST MF) with up to three predicted parameter matches from the QSPR models to generate a combined CASI Score representing the measure of confidence for the identification. Threshold values were applied to the CASI Scores assigned to proposed structures, which improved the accuracy for the classification of true/false positives and true/false negatives. Results for the identification of compounds have been validated, and it has been demonstrated that identification using CASI is more accurate than using NIST MS Search alone. CASI is an easily accessible web-interfaced software platform which represents an innovative, high-throughput system that allows fast and accurate identification of constituents in complex matrices, such as those requiring 2D separation techniques.

Improved coarse-grained model for molecular-dynamics simulations of water nucleation.

We developed a new coarse-grained (CG) model for water to study nucleation of droplets from the vapor phase. The resulting potential has a more flexible functional form and a longer range cutoff compared to other CG potentials available for water. This allowed us to extend the range of applicability of coarse-grained techniques to nucleation phenomena. By improving the description of the interactions between water molecules in the gas phase, we obtained CG model that gives similar results than the all-atom (AA) TIP4P model but at a lower computational cost. In this work we present the validation of the potential and its application to the study of nucleation of water droplets from the supersaturated vapor phase via molecular-dynamics simulations. The computed nucleation rates at T = 320 K and 350 K at different supersaturations, ranging from 5 to 15, compare very well with AA TIP4P simulations and show the right dependence on the temperature compared with available experimental data. To help comparison with the experiments, we explored in detail the different ways to control the temperature and the effects on nucleation.

Mechanisms of propylene glycol and triacetin pyrolysis.

Propylene glycol and triacetin are chemical compounds, commonly used as food additives. Though the usage of the pure chemicals is not considered harmful when used as dietary supplements, little is known about the nature of their thermal degradation products and the impact they may have on human health. For these reasons, in this manuscript we investigate the thermal decomposition mechanisms of both neutral propylene glycol and triacetin in the gas phase by a novel simulation framework. This is based on a free energy sampling methodology followed by an accurate energy refinement. Structures, Gibbs free energy barriers, and rate constants at 800 K were computed for the different steps involved in the two pyrolytic processes. The thermal decomposition mechanisms found theoretically for propylene glycol and triacetin were validated by a qualitative experimental investigation using gas-phase chromatography-mass spectroscopy, with excellent agreement. The results provide a validation of the novel simulation framework and shed light on the potential hazard to the health that propylene glycol and triacetin may have when exposed to high temperatures.

Toxicological Assessment of Flavor Ingredients in E-Vapor Products.

Many flavor ingredients are often used in potentially reduced-risk tobacco products (such as e-vapor products). Although most are "generally recognized as safe (GRAS)" when used in food, there is limited information available on their long-term health effects when delivered by inhalation. While obtaining route-of-exposure-specific toxicological data on flavor ingredients is critical to product evaluation, the large number of individual flavor ingredients available and their potential combinations render classical toxicological assessment approaches impractical, as they may require years of preclinical investigations and thousands of laboratory animals. Therefore, we propose a pragmatic approach in which flavor ingredients are initially assigned to groups of structurally related compounds (Flavor Groups), from which flavor group representatives (FGR) are then selected and tested individually and as a mixture in vitro and in vivo. The premise is that structurally related compounds would have comparable metabolic and biological activity and that the data generated using FGRs could support the toxicological assessment of other structurally related flavor ingredients of their respective Flavor Groups. This approach is explained in a step-wise manner and exemplified by a case study, along with its strengths, limitations as well as recommendations for further confirmatory testing. Once completed, this FGR approach could significantly reduce the time and resources required for filling the data gap in understanding the health risks of many flavor ingredients while also minimizing the need for laboratory animals.

Design, synthesis, and functional evaluation of leukocyte function associated antigen-1 antagonists in early and late stages of cancer development.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its α(L)-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.

Building an R&D chemical registration system.

Small molecule chemistry is of central importance to a number of R&D companies in diverse areas such as the pharmaceutical, nutraceutical, food flavoring, and cosmeceutical industries. In order to store and manage thousands of chemical compounds in such an environment, we have built a state-of-the-art master chemical database with unique structure identifiers. Here, we present the concept and methodology we used to build the system that we call the Unique Compound Database (UCD). In the UCD, each molecule is registered only once (uniqueness), structures with alternative representations are entered in a uniform way (normalization), and the chemical structure drawings are recognizable to chemists and to a cartridge. In brief, structural molecules are entered as neutral entities which can be associated with a salt. The salts are listed in a dictionary and bound to the molecule with the appropriate stoichiometric coefficient in an entity called "substance". The substances are associated with batches. Once a molecule is registered, some properties (e.g., ADMET prediction, IUPAC name, chemical properties) are calculated automatically. The UCD has both automated and manual data controls. Moreover, the UCD concept enables the management of user errors in the structure entry by reassigning or archiving the batches. It also allows updating of the records to include newly discovered properties of individual structures. As our research spans a wide variety of scientific fields, the database enables registration of mixtures of compounds, enantiomers, tautomers, and compounds with unknown stereochemistries.

Molecular basis for Bcl-2 homology 3 domain recognition in the Bcl-2 protein family: identification of conserved hot spot interactions.

The proteins of the Bcl-2 family are important regulators of apoptosis, or programmed cell death. These proteins regulate this fundamental biological process via the formation of heterodimers involving both pro- and anti-apoptotic family members. Disruption of the balance between anti- and pro-apoptotic Bcl-2 proteins is the cause of numerous pathologies. Bcl-xl, an anti-apoptotic protein of this family, is known to form heterodimers with multiple pro-apoptotic proteins, such as Bad, Bim, Bak, and Bid. To elucidate the molecular basis of this recognition process, we used molecular dynamics simulations coupled with the Molecular Mechanics/Poisson-Boltzmann Surface Area approach to identify the amino acids that make significant energetic contributions to the binding free energy of four complexes formed between Bcl-xl and pro-apoptotic Bcl-2 homology 3 peptides. A fifth protein-peptide complex composed of another anti-apoptotic protein, Bcl-w, in complex with the peptide from Bim was also studied. The results identified amino acids of both the anti-apoptotic proteins as well as the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic proteins that make strong, recurrent interactions in the protein complexes. The calculations show that the two anti-apoptotic proteins, Bcl-xl and Bcl-w, share a similar recognition mechanism. Our results provide insight into the molecular basis for the promiscuous nature of this molecular recognition process by members of the Bcl-2 protein family. These amino acids could be targeted in the design of new mimetics that serve as scaffolds for new antitumoral molecules.

Comparative normal mode analysis of LFA-1 integrin I-domains.

The conformational dynamics of the Inserted domain (I-domain) from the lymphocyte function-associated antigen-1 (LFA-1) was investigated by normal mode analysis of multiple structures of the low, intermediate, and high affinity states. LFA-1 is an integrin expressed on leukocytes and is of critical importance in adhesion reactions, like antigen-specific responses, homing, and diapedesis. The main ligand binding site of LFA-1 is the I-domain, which recognizes intercellular adhesion molecules (ICAMs), members of the immunoglobulin superfamily. From experimental crystal structures, a large-scale conformational change of, among others, the alpha7 helix of the I-domain has been observed leading to the proposal that these structural changes are linked to the conformational regulation of LFA-1. The results from the present calculations show that structural changes of the alpha7 helix consistent with those observed in the crystal structures are significantly sampled by the low frequency modes. This was found to be particularly true for the low affinity state of the I-domain, indicating that low frequency motions favor the conformational transition implicated in activation. However, beyond the simple downward shift of the helix implied by the crystal structures, the calculations further show that there is a noticeable swinging-out motion of the helix. The consequences of this motion are discussed in the context of integrin activation and inhibition. Moreover, significant changes in the atomic-level dynamics and in long-range correlated motions of the I-domain were found to occur upon binding of the natural ligand ICAM. These changes were more local upon binding of an allosteric inhibitor. The present study opens the question of how changes in dynamics may contribute to the long-range transmission of signal upon ICAM binding by the LFA-1 I-domain.

Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor.

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

Dual specificity of the interfacial inhibitor brefeldin a for arf proteins and sec7 domains.

Guanine nucleotide exchange factors (GEFs), which activate small GTP-binding proteins (SMG) by stimulating their GDP/GTP exchange, are emerging as candidate targets for the inhibition of cellular pathways involved in diseases. However, their specific inhibition by competitive inhibitors is challenging, because GEF and SMG families comprise highly similar members. Nature shows us an alternative strategy called interfacial inhibition, exemplified by Brefeldin A (BFA). BFA inhibits the activation of Arf1 by its GEFs in vivo by stabilizing an abortive complex between Arf-GDP and the catalytic Sec7 domain of some of its GEFs. Here we characterize the specificity of BFA toward wild-type (ARNO and BIG1) and mutant Sec7 domains and toward class I, II, and III Arfs. We find that BFA sensitivity of the exchange reaction depends on the nature of both the Sec7 domain and the Arf protein. A single Phe/Tyr substitution is sufficient to achieve BFA sensitivity of the Sec7 domain, which is supported by our characterization of brefeldin C (BFC), a BFA analog that cannot interact with the Tyr residue, and by free energy computations. We further show that Arf1 and Arf5, but not Arf6, are BFA-sensitive, despite their having every BFA-interacting residue in common. Analysis of Arf6 mutants points to the dynamics of the interswitch, which is involved in membrane-to-nucleotide signal propagation, as contributing to, although not sufficient for, BFA sensitivity. Altogether, our results reveal the Tyr/Phe substitution as a novel tool for monitoring BFA sensitivity of cellular ArfGEFs and document the exquisite and dual specificity that can be achieved by an interfacial inhibitor.