Minireview PHARMACOPHORE AND THREE-DIMENSIONAL QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP METHODS FOR MODELING CYTOCHROME P450 ACTIVE SITES

This paper is available online at http://dmd.aspetjournals.org ABSTRACT: Structure activity relationships (SAR), three-dimensional structure activity relationships (3D-QSAR), and pharmacophores represent useful tools in understanding cytochrome P450 (CYP) active sites in the absence of crystal structures for these human enzymes. These approaches have developed over the last 30 years such that they are now being applied in numerous industrial and academic laboratories solely for this purpose. Such computational approaches have helped in understanding substrate and inhibitor binding to the major human CYPs 1A2, 2B6, 2C9, 2D6, 3A4 as well as other CYPs and additionally complement homology models for these enzymes. Similarly, these approaches may assist in our understanding of CYP induction. This review describes in detail the development of pharmacophores and 3D-QSAR techniques, which are now being more widely used for modeling CYPs; the review will also describe how such approaches are likely to further impact our active site knowledge of these omnipresent and important enzymes. By the end of the 1990s, several reviews had characterized the active site details and physicochemical properties of substrates for the major cytochrome P450 (CYP 1 ) enzymes. These reviews had been gathered from analysis of physicochemical data 1 Abbreviations used are: CYP or P450, cytochrome P450; CoMFA, comparative molecular field analysis; GOLPE, generating optimal linear PLS estimations; PLS, partial least squares; 3D-QSAR, three-dimensional quantitative structure-activity relationship; MS-WHIM, molecular surface weighted holistic invariant molecular. DMD 29:936-944, 2001 Printed in U.S.A. 936 intends to give an overview of the pharmacophore and 3D-QSAR models that have been used to describe P450s and indicate their varying degrees of success. 3D-QSAR and Pharmacophores The development of computational tools has paralleled that of in vitro approaches to understanding and characterizing CYPs. One of the first visual 3D-QSAR computational approaches was comparative molecular field analysis (CoMFA) Until recently, few CYP binding or active site models had been generated using enzyme kinetic data, and these focused primarily on inhibition. Now, however, a considerable number of CYP pharmacophores have appeared in the literature, which presents us with the opportunity to review what is known about several CYPs based on such computational analyses. CYP Models CYP1A2. CYP1A2 is an inducible member of the CYP superfamily, which can be inhibited by some selective serotonin reuptake inhibitors With regard to predicting substrates for CYP1A2, one study has suggested that they are generally neutral or protonated and that they possess a total interaction energy greater than Ϫ40 kcal/mol and a molecular volume lower than 200 Å 3 CYP2A6. To date there has been no published CYP2A6 QSAR; however, the related mouse form, CYP2A5, has been studied. One group analyzed substrate requirements using a graphical method and concluded that bicyclic ring systems with an electron-rich moiety were essential for the 11 molecules analyzed CYP2B6. Many examples of xenobiotics metabolized in part by CYP2B6 have been identified and described in more detail This challenge was answered by The first quantitative QSAR for CYP2C9 was published in 1996 This model was validated by testing 14 new compounds that had K i values ranging from 0.1 to 48 M (Rao et al., 2000). While the initial training set contained mostly coumarin-containing compounds, this validation set contained mostly sulfonamides. Interestingly, the initial model predicted the affinity of the validation set reasonably well, predicting 13 of the 14 compounds within 1 log residual. Finally, when these compounds where included in the training set, the pharmacophore remained essentially the same. In separate experiments, conducted at the same time as the validation study described above, pharmacophore and PLS predictive models where constructed using Catalyst and PLS MS-WHIM, respectively To gain confidence in the pharmacophores generated for CYP2C9, an attempt was made to determine the specific amino acid residues that might be involved in establishing the pharmacophore. Initial docking of the 9(R)-11(S)-cyclocoumarol and visualizing the CoMFA field in a CYP2C9 homology model indicated that two phenylalanine residues, Phe 110 EKINS ET AL CYP2C19. One group has focused on obtaining substrate structure activity relationships for the polymorphic CYP2C19 using inhibitors of omeprazole 5-hydroxylation (Lock et al., 1998a,b). Using mainly benzodiazepines which are N-dealkylated and 3-hydroxylated, it was suggested that these sites and the carbonyl group were important for inhibition. Electron-withdrawing groups were found to further decrease inhibition. As yet, the data for the 14 compounds used in these two studies have not been used to produce a published 3D-QSAR. CYP2D6. Human CYP2D6 is a polymorphic member of the CYP superfamily and is absent in 5 to 9% of the Caucasian population as a result of a recessive inheritance of gene mutations The first substrate models were manual alignments based on substrates containing a basic nitrogen atom at either 5 Å Another small-molecule model for CYP2D6 was derived by The actual positions of the heme moiety and the I-helix containing Asp 301 [derived from a protein homology model of CYP2D6 Recently, a combined pharmacophore and homology model for CYP2D6 has been derived (de Groot et al., 1999a,b). This model consists of a set of two pharmacophores (one for O-dealkylation and oxidation reactions and a second one for N-dealkylation reactions catalyzed by CYP2D6) embedded in a protein homology model based on bacterial CYP crystal structures (de Groot et al., 1999a,b). This model for the first time combines the strengths of pharmacophore models (atom-atom overlap and reproducible starting points) and homology models (steric interactions and the possibility to identify amino acids involved in binding). This model correctly predicted the metabolism of a wide variety of compounds (de Groot et al., 1999a,b). An inhibitor model for CYP2D6 has also been derived. The template of this model was derived by fitting six strong reversible inhibitors of CYP2D6 onto each other . The basic nitrogen atoms were superimposed and the aromatic planes of these inhibitors were fitted coplanar. Consecutively, other inhibitors, such as derivatives of ajmalicine and quinidine, were fitted onto the derived template. The derived preliminary pharmacophore model consisted of a tertiary nitrogen atom (protonated at physiological pH) and a flat hydrophobic region. There also appeared to be two regions in which functional groups with lone pairs were allowed. In one of these regions, these groups caused enhanced inhibitory potency, which was not the case in yet another region . The overall criteria derived for this inhibitor-based small-molecule model were very similar to the criteria for the proposed substrate models of CYP2D6 P450 PHARMACOPHORE AND QSAR MODELS A set of 3D/4D-QSAR pharmacophore models has also been created for competitive inhibitors of CYP2D6 in a manner similar to that described for CYP2B6 and CYP2C9 using Catalyst CYP2E1. CYP2E1 is involved in the metabolism of many toxic and carcinogenic molecules such as low molecular weight solvents and anesthetics. Early on, it was suggested that the active site was restricted due to the limited size of known substrates. A graphical model of the active site topology was derived from reactions of human CYP2E1 with phenyldiazene, 2-naphthyl, and p-biphenylhydrazine. This work indicated that the active site was open above the pyrrole rings A and D of the heme for a height of 10 Å CYP3A4. Smith et al. have described in detail the CYP3A4 active site characteristics (as well as those of the other major mammalian CYPs) based on homology models built using soluble bacterial CYP structures as a template More recently, a pharmacophore for inhibitors of CYP3A4-mediated midazolam 1Ј-hydroxylase was developed that consisted of four features necessary for the inhibition of CYP3A4 To evaluate these 3D-QSAR models, the activity of molecules excluded from the training set was predicted and then compared with those observed by means of a 1 log residual. Eight molecules were selected from the literature with K i (apparent) values. Both of the CYP3A4 K i (apparent) Catalyst models predicted the K i values similarly. Seven of eight best fit predictions were within 1 log unit residual, for both models, and the correct rank ordering of three protease inhibitors was observed Using the same commercially available software, a Catalyst hypothesis for 38 CYP3A4 substrates was generated using literature K m data Analyses of the likely features of activators of CYP3A4 have also been undertaken, as three substrates (carbamazepine, nifedipine, and testosterone) within the 38-molecule training set used in the CYP3A4 pharmacophore were known CYP3A4 autoactivators. A common features analysis of these molecules using the HipHop function within Catalyst generated a pharmacophore illustrating three hydrophobic areas and one hydrogen bond acceptor. The hydrophobic areas were located 4.4 to 7.6 Å from the hydrogen bond acceptor feature, and the sites of metabolism were colocated. Therefore, hydrophobic interactions with the CYP3A4 active site may be more important than hydrogen bonding for these same CYP3A4 substrates CYP19 (Aromatase). The importance of CYPs that metabolize endogenous substrates can be demonstrated by aromatase, which catalyzes the metabolism of androstenedione to estrone, 16␣-hy- EKINS ET AL droxyandrostenedione to estriol, and testosterone to estradiol via the aromatization of the A ring and the removal of the C19 methyl group CYP51 (14␣-Demethylase)

Sport participation after the HandbikeBattle:benefits, barriers, facilitators from the event-a follow-up survey

OBJECTIVES: To investigate sports participation among individuals in the HandbikeBattle project 1.5–5.5 years after the event, the barriers and facilitators to sport participation and benefits of participating in the event. SETTING: Former participants of the HandbikeBattle, a handcycling race on an Austrian mountain. METHODS: Handcyclists who took part in one or more HandbikeBattle events in 2013–2017 were invited to complete a survey in December 2018. Questions were asked on benefits of participating in this event, current sport participation, and experienced barriers and facilitators regarding current sport participation. RESULTS: Respondents (N = 96 (N = 59 with spinal cord injury (SCI) or Spina Bifida (SB)), response rate = 47%) reported benefits from this event regarding their fitness level (90%), personal development (81%), daily life activities (66%), and health (64%). Median current sport participation was 5 h/week (IQR: 3–8). Most frequently indicated personal barriers for sports participation were: lack of time (31%), disability (17%), and pain (15%). Most frequently mentioned environmental barriers were: transportation time to sport accommodation (19%), and lack of peers to do sports with (16%). Most important facilitators were motivation to improve health and/or fitness (92%), fun and/or relaxation (85%). The results for the subgroup with SCI were comparable with the results of the total group. CONCLUSIONS: Results highlight the role sports challenges can have in establishing a sustainable active life lifestyle among people with disabilities

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines

Advanced glycation endproducts are increased in rheumatoid arthritis patients with controlled disease

Introduction: Advanced glycation end products (AGEs) are produced and can accumulate during chronic inflammation, as might be present in patients with rheumatoid arthritis (RA). AGEs are involved in the development of cardiovascular disease. The aim of this study is to evaluate whether AGEs are increased in patients with long-standing RA and whether AGE accumulation is related to disease activity, disease severity and measures of (premature) atherosclerosis, such as endothelial activation, endothelial dysfunction and intima media thickness (IMT). Methods: In a cross-sectional study, 49 consecutive RA patients with longstanding disease (median disease duration of 12.3 years (range 9.3 to 15.1)), receiving standard of care, were included and compared with 49 age-and sex-matched healthy controls (HC). AGEs were determined by skin autofluorescence. Disease activity was evaluated by the Disease Activity Score of 28 joints (DAS-28) score and joint damage by modified Sharp-v.d. Heijde score. Endothelial activation (soluble vascular cellular adhesion molecule-1) sVCAM-1, von Willebrand factor (vWF), thrombomodulin), endothelial dysfunction (determined by small artery elasticity (SAE)) and IMT were measured and related to AGE accumulation. Results: AGEs were increased in RA patients (median 2.4 arbitrary units (a.u.), range 1.6 to 4.2) compared to HC (2.2, 1.3 to 3.8). RA patients had a DAS-28 score of 2.9 (0.8 to 6.9) and a modified Sharp-v.d. Heijde score of 19 (0 to 103). sVCAM-1 and vWF levels were higher in RA patients. SAE was significantly decreased in RA (3.9 ml/mmHg (1.4 to 12.2) vs. 6.1 in HC (1.7 to 12.9). IMT did not differ between the two groups. Combining both groups' AGEs correlated with vWF, sVCAM-1 and IMT, and was inversely related to SAE. In RA, AGEs had an inverse relation with SAE, but did not relate to disease activity or radiological damage. In multivariate analysis for both groups, smoking, glucose levels, vWF, SAE and male gender were significantly related to the formation of AGEs. Conclusions: AGEs were increased in RA patients with long-standing disease and without signs of premature atherosclerosis. AGEs were related to endothelial activation and endothelial dysfunction. This supports the hypothesis that in RA AGEs may be an early marker of cardiovascular disease

The Course of Physical Capacity in Wheelchair Users During Training for the HandbikeBattle and at 1-Yr Follow-up

OBJECTIVE: The aims of this study were (1) to compare physical capacity at 1-yr follow-up with physical capacity before and after the training period for the HandbikeBattle event and (2) to identify determinants of the course of physical capacity during follow-up. DESIGN: This was a prospective observational study. Former rehabilitation patients (N = 33) with health conditions such as spinal cord injury or amputation were included. A handcycling/arm crank graded exercise test was performed before (January, T1) and after the training period (June, T2) and at 1-yr follow-up (June, T4). Outcomes were peak power output (W) and peak oxygen uptake (L/min). Determinants were sex (male/female); age (years); classification; physical capacity, musculoskeletal pain, exercise stage of change, and exercise self-efficacy at T1; and HandbikeBattle participation at T4. RESULTS: Multilevel regression analyses showed that peak power output and peak oxygen uptake increased during the training period and did not significantly change during follow-up (T1: 112 ± 37 W, 1.70 ± 0.48 L/min; T2: 130 ± 40 W, 2.07 ± 0.59 L/min; T4: 126 ± 42 W, 2.00 ± 0.57 L/min). Participants who competed again in the HandbikeBattle showed slight improvement in physical capacity during follow-up, whereas participants who did not compete again showed a decrease. CONCLUSION: Physical capacity showed an increase during the training period and remained stable after 1-yr follow-up. Being (repeatedly) committed to a challenge might facilitate long-term exercise maintenance