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)

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

Low drop-out rates in the HandbikeBattle free-living training study:understanding the reasons for dropping out

STUDY DESIGN: Longitudinal observational study. OBJECTIVES: During the five-month free-living training period for the HandbikeBattle event several participants dropped out. The aim of this study was to clarify the numbers and reasons for drop out, and to characterize the differences between study participants who did (dropouts) and did not (competitors) drop out during the training period for the HandbikeBattle event. SETTING: Former participants of the HandbikeBattle, a handcycling race on an Austrian mountain. METHODS: Participants (N = 313 (N = 209 (67%) with spinal cord injury or spina bifida)) enrolled between 2013-2018. Drop out and reasons for drop out were registered. Competitors and dropouts were compared regarding personal, disability, physical, and psychological factors, which were measured at the start of the training period. RESULTS: Forty-five participants (14%) dropped out during the training period with medical complications (49%) and motivational problems (29%) as main reasons. The only differences were that competitors participated more in sports before the study (p = 0.01) and achieved a higher peak power output (p = 0.04) compared to dropouts. CONCLUSIONS: The drop-out rate of the HandbikeBattle study was low compared to previous exercise intervention studies, which might be related to the less strictly imposed free-living training. Persons with less experience in sport and a lower fitness level might need more attention during a training intervention to prevent them from dropping out

Training for the HandbikeBattle:an explorative analysis of training load and handcycling physical capacity in recreationally active wheelchair users

Purpose: (1) to analyze training characteristics of recreationally active wheelchair users during handcycle training, and (2) to examine the associations between training load and change in physical capacity. Methods: Former rehabilitation patients (N = 60) with health conditions such as spinal cord injury or amputation were included. Participants trained for five months. A handcycling/arm crank graded exercise test was performed before and after the training period. Outcomes: peak power output per kg (POpeak/kg) and peak oxygen uptake per kg (VO 2peak/kg). Training load was defined as Training Impulse (TRIMP), which is rating of perceived exertion (sRPE) multiplied by duration of the session, in arbitrary units (AU). Training intensity distribution (TID) was also determined (time in zone 1, RPE ≤4; zone 2, RPE 5–6; zone 3, RPE ≥7). Results: Multilevel regression analyses showed that TRIMP sRPE was not significantly associated with change in physical capacity. Time in zone 2 (RPE 5–6) was significantly associated with ΔVO 2peak, %ΔVO 2peak, ΔVO 2peak/kg and %ΔVO 2peak/kg. Conclusion: Training at RPE 5–6 was the only determinant that was significantly associated with improvement in physical capacity. Additional controlled studies are necessary to demonstrate causality and gather more information about its usefulness, and optimal handcycle training regimes for recreationally active wheelchair users.IMPLICATIONS FOR REHABILITATION Monitoring of handcycle training load is important to structure the training effort and intensity over time and to eventually optimize performance capacity. This is especially important for relatively untrained wheelchair users, who have a low physical capacity and a high risk of overuse injuries and shoulder pain. Training load can be easily calculated by multiplying the intensity of the training (RPE 0–10) with the duration of the training in minutes. Results on handcycle training at RPE 5–6 intensity in recreationally active wheelchair users suggests to be promising and should be further investigated with controlled studies

Secondary health conditions in persons with a spinal cord injury for at least 10 years:design of a comprehensive long-term cross-sectional study

Purpose: To describe the prevalence of secondary health conditions (SHCs) (urinary tract and bowel problems, pressure ulcers, spasticity, musculoskeletal and neuropathic pain, sexual dysfunction, respiratory and cardiovascular disorders) in persons with long-term spinal cord injury (SCI), and to explore the impact of SHCs on fitness, active lifestyle, participation and well-being. Methods: A time since injury (TSI)-stratified cros-ssectional study among 300 persons between 28- and 65-year-old with a SCI for at least 10 years. Strata of TSI are 10-19, 20-29, and 30 or more years. All eight Dutch rehabilitation centres with a SCI unit will participate. Participants will be invited for a 1-day visit to the rehabilitation centre for an aftercare check-up by the local SCI rehabilitation physician (neurological impairment, SHCs and management), physical tests by a trained research assistant (lung function, wheelchair skills, physical capacity), and they will be asked to complete a self-report questionnaire in advance. Results: Not applicable. Conclusion: This study will provide knowledge on the health status and functioning of persons aging with SCI living in the Netherlands. This knowledge will help us to develop predictive models for the occurrence of SHCs and to formulate guidelines to improve health care for persons with long-term SCI.</p

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas

Evolution of mitral regurgitation in patients with heart failure referred to a tertiary heart failure clinic

Aims: Significant mitral regurgitation (MR) is an important predictor for all-cause mortality and heart failure (HF) hospitalizations independent of left ventricular ejection fraction (LVEF). The aims of this study were to investigate (i) in how many patients referred to a tertiary outpatient HF clinic HF therapy could be optimized, (ii) the effect of optimized treatment on MR severity, and (iii) whether a reduction in MR resulted in improvement of symptoms. Methods and results: Forty-seven referred patients with therapy-resistant symptomatic chronic HF with an LVEF <40% and at least moderate MR were analysed on admission and after optimization of HF treatment after 6–18 months. The patients were classified as a volume responder when LV end-systolic volume (LVESV) decreased ≥15%, as LVEF responder when LVEF increased by ≥5% points, as clinical responder when New York Heart Association (NYHA) class improved at least one category, and as MR responder when MR severity improved at least one category to maximally moderate. After 14 ± 4 months of treatment optimization, optimal doses of angiotensin-converting enzyme inhibitors/angiotensin receptor blocker were seen in 18 (38%) patients compared with three (6%) at baseline (P < 0.001), and optimal doses of beta-blockers were seen in 14 (30%) patients compared with four (9%) at baseline (P < 0.001). In total, 68% of the patients were clinical responders, 57% MR responders, 34% volumetric responders, and 49% LVEF responders. NYHA class improved from 2.9 ± 0.6 to 2.0 ± 0.9 (P < 0.001), MR class from 5.2 ± 0.8 to 3.6 ± 1.5 (P < 0.001), LVEF from 24% ± 9% to 31% ± 12% (P < 0.01), and LVESV non-significantly improved. The positive predictive value of MR response to NYHA response was 88%; the negative predictive value was 53%, agreement 69%, and kappa 0.39. The positive predictive value of LVEF response to NYHA response was 76%; the negative predictive value was 44%, agreement 60%, and kappa 0.21. The positive predictive value of LVESV volume response to NYHA response was 75%; the negative predictive value was 39%, agreement 51%, and kappa 0.12. Conclusions: Although this study was limited by a small number of patients, initiation and up-titration of recommended HF therapy in patients referred to our tertiary HF outpatient clinic resulted in significant MR reduction in over half of the patients, emphasizing the importance of optimal medical treatment in these very sick cardiac patients with otherwise grave prognosis. MR reduction was best correlated to NYHA improvement