Development and Application of a Novel Enhanced Sampling Method and Bayesian Analysis for Characterizing Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are a class of proteins with wide-rangingsignificance in signaling and disease that do not adopt a dominant folded structure asmonomers. Rather, the structures of IDPs in solution are best described as ensembles ofconformational states that may range from being fully random coil to partially ordered.This structural plasticity of IDPs is theorized to facilitate regulation of their interactionwith other species, as in signal transduction or aggregation of IDPs into ordered fibrils.Characterizing the structural ensembles of IDPs in the free, solvated state is key tounderstanding the mechanisms of these interactions, and correspondingly the role an IDPspecies plays in signaling or disease.The rapid interconversion between conformational states, however, complicates theexperimental study of IDPs because most experimental signals report highly averagedinformation. Computational modeling with validation through comparison to experimenthas therefore been a main approach to characterizing IDP structure and dynamics. Thefocus of my dissertation is on the development of new methods for computational study ofIDPs, facilitating better and less expensive de novo generation of IDP structural ensemblesand improving the metrics used to evaluate the degree of agreement between a simulatedensemble and a set of experimental data.Despite vast improvements in computational power and efficiency, moleculardynamics (MD) simulations of IDPs for generating conformational ensembles are stilllimited by the expense of calculations. In Chapter 2 I present the development of a newenhanced sampling method – temperature cool walking (TCW) – and comparison of itsperformance against a standard method – temperature replica exchange (TREx). The TCWmethod accelerates the rate of convergence to the equilibrium conformational ensemblewith increased sampling acceleration relative to TREx at greatly reduced computationalcost.The second major limitation in MD is the accuracy of the force field. Most classicalfixed charge force fields were parameterized using data from folded proteins, and havebeen thought to be biased to overly collapsed and structured conformations. This hasmotivated the development of IDP-tailored force fields that sample greater disorder, at thepotential expense of the ability to model stabilizing interactions between an IDP and itsbinding partners. In Chapter 3, I assess to what degree the shortcomings assigned tostandard force fields may be due to insufficient sampling by characterizing theperformance of standard and newly modified force fields on the Alzheimer’s peptideamyloid-β using both TREx and TCW. We find that with improved sampling, standard andmodified force fields produce similar structural ensembles, suggesting that both areappropriate for simulation of the disordered state. In Chapter 4 I present preliminaryresults building off of this work by characterizing the performance of a polarizable forcefield modeling a synthetic peptide that demonstrates complete loss of helical content withincreasing temperature. Inclusion of polarization effects has been thought to be key foraccurate modeling of such multicomponent systems, especially when there is a shift in theelectrostatic environment as is the case for the unfolding peptide. Our early results, whilelimited by current lack of convergence for tests using the polarizable force field andneeding further confirmation, match that expectation by finding early evidence of greaterresponse to temperature by the polarizable force field than fixed charge comparators.The last work presented here is in the development of new methods for calculatingthe degree of agreement between a simulated IDP ensemble and experimental data. Backcalculationof experimental data from structure can be very imprecise, motivating thedevelopment in Chapter 5 of scoring formalisms that account for variable uncertainties inboth back-calculation and experiment for diverse experimental data types. In summary, themethods described in this dissertation seek to improve computational study of IDPs byfacilitating better, less expensive generation of IDP ensembles and producing moreinformative metrics for evaluating their agreement with experiment

Abrupt vessel closure complicating coronary angioplasty: Clinical, angiographic and therapeutic profile

AbstractTo assess the clinical, angiographic and procedural correlates of outcome after abrupt vessel closure during coronary angioplasty, results were analyzed of 109 patients (8.3%) who had abrupt vessel closure during 1,319 consecutive coronary angioplasty procedures performed between July 1, 1988 and June 30, 1990. These 109 patients had a mean age of 59 ± 11 years; 63% were male, 57% had had a prior myocardial infarction and 61% had multivessel disease. Coronary angioplasty was performed in the settings of acute myocardial infarction (14%), recent myocardial infarction (36%), unstable angina (34%) and stable ischemia (29%).Abrupt vessel closure occurred at a median of 27 min (range 0 min to 5 days) from the first balloon inflation. By angiographic criteria, thrombus or coronary dissection was identified in 20% and 28% of cases, respectively; both thrombus and dissection were present in 7% of closures, and 45% were due to indeterminate mechanisms. Successful reversal of abrupt vessel closure, defined as restoration of normal Thrombolysis In Myocardial Infarction (TIMI) grade 3 flow without resultant Q wave myocardial infarction, emergency bypass surgery or death, was achieved in 47 patients (43%). By hierarchal analysis, the incidence of death, emergency coronary bypass surgery, Q wave and non-Q wave myocardial infarction was 8%, 20%, 9% and 11%, respectively.Univariate analysis using 23 clinical, morphologic and procedural variables demonstrated that successful outcome after abrupt closure was associated with prolonged balloon inflations (>120 s) (odds ratio = 6.87, p < 0.001), unstable angina (odds ratio = 2.37, p = 0.034) and placement of an intracoronary stent (odds ratio = 5.33, p = 0.062). By multivariate analysis, independent correlates of successful outcome were prolonged balloon inflations (odds ratio = 5.11, p = 0.001) and intracoronary stenting (odds ratio = 4.37, p = 0.049).Thus, although prolonged balloon inflations and intracoronary stents may improve outcome after abrupt vessel closure, the cumulative risk of morbidity or mortality remains significant and mandates investigation into improved strategies for its prevention and treatment

The duration of pretreatment with ticlopidine prior to stenting is associated with the risk of procedure-related non–Q-wave myocardial infarctions

AbstractObjectives. This study sought to determine whether the duration of pretreatment with the adenosine diphosphate receptor antagonist ticlopidine prior to intracoronary stenting is associated with the incidence of procedure-related non–Q-wave myocardial infarctions (MIs).Background. Dual antiplatelet therapy with ticlopidine and aspirin is routinely used with stenting, although ticlopidine is commonly not begun until the day of the procedure. Periprocedural MIs are at least partially platelet-dependent events. As the maximal platelet inhibitory effects of this drug take 2 to 3 days to be realized, we hypothesized that longer treatment prior to stenting would be associated with lower rates of procedure-related MIs.Methods. We reviewed outcomes in 175 consecutive patients treated with ticlopidine prior to stenting at the Cleveland Clinic Foundation. Those patients with an elevation in creatine kinase above our laboratory normal (>210 IU/L) with ≥4% MB fraction on routine evaluation were defined as having a non–Q-wave MI.Results. There were 28 patients (16%) who had a non–Q-wave MI. Longer duration of ticlopidine pretreatment was strongly associated with a lower incidence of procedure-related non–Q-wave MIs (duration of pretreatment <1 day, 29% had MI; 1 to 2 days, 14%; ≥3 days, 5%; chi-square for trend = 9.6; p = 0.002). Ticlopidine pretreatment of ≥3 days was associated with a significant reduction in the risk of non–Q-wave MI (unadjusted odds ratio 0.18, 95% confidence interval = 0.04 to 0.78, p = 0.01) compared with pretreatment of <3 days.Conclusions. Among patients undergoing intracoronary stenting, beginning ticlopidine therapy several days prior to the procedure is associated with a reduced risk of procedural non–Q-wave MIs

Evidence for Prevention of Death and Myocardial Infarction With Platelet Membrane Glycoprotein IIb/IIIa Receptor Blockade by Abciximab (c7E3 Fab) Among Patients With Unstable Angina Undergoing Percutaneous Coronary Revascularization fn1fn1This study was supported by Centocor, Inc., Malvern, Pennsylvania.

AbstractObjectives. We sought to evaluate whether patients with unstable angina undergoing coronary intervention derive particular clinical benefit from potent platelet inhibition.Background. Plaque rupture and platelet aggregation are pathogenetic processes common to unstable angina and ischemic complications of percutaneous coronary intervention.Methods. Of the 2,099 patients undergoing a coronary intervention in the Evaluation of 7E3 in Preventing Ischemic Complications (EPIC) trial, 489 were enrolled with the diagnosis of unstable angina and randomized to receive placebo, an abciximab (c7E3) bolus immediately before the intervention or an abciximab bolus followed by a 12-h infusion. The primary end point was a composite of death, myocardial infarction (MI) or urgent repeat revascularization within 30 days of randomization. The occurrence of death, MI or any revascularization within 6 months was also assessed.Results. Compared with placebo, the bolus and infusion of abciximab resulted in a 62% reduction in the rate of the primary end point (12.8% vs. 4.8%, p = 0.012) among patients with unstable angina, due primarily to a reduction in the incidences of death (3.2% vs. 1.2%, p = 0.164) and MI (9% vs. 1.8%, p = 0.004). By 6 months, cumulative death and MI were further reduced by abciximab (6.6% vs. 1.8%, p = 0.018 and 11.1% vs. 2.4%, p = 0.002, respectively). The magnitude of the risk reduction with abciximab was greater among the patients with unstable angina than among other patients in the EPIC trial without unstable angina for the end points of death (interaction: p = 0.008 at 30 days, p = 0.002 at 6 months) and MI (interaction: p = 0.004 at 30 days, p = 0.003 at 6 months).Conclusions. The syndrome of unstable angina identifies patients who will experience particularly marked reductions in the risk of death and MI with abciximab during coronary intervention.(J Am Coll Cardiol 1997;30:149–56

Association of Lowering Low‐Density Lipoprotein Cholesterol With Contemporary Lipid‐Lowering Therapies and Risk of Diabetes Mellitus: A Systematic Review and Meta‐Analysis

Background The relationship between lowering LDL (low‐density lipoprotein) cholesterol with contemporary lipid‐lowering therapies and incident diabetes mellitus (DM) remains uncertain. Methods and Results Thirty‐three randomized controlled trials (21 of statins, 12 of PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitors, and 0 of ezetimibe) were selected using Medline, Embase, and the Cochrane Central Register of Controlled Trials (inception through November 15, 2018). A total of 163 688 nondiabetic patients were randomly assigned to more intensive (83 123 patients) or less intensive (80 565 patients) lipid‐lowering therapy. More intensive lipid‐lowering therapy was defined as the more potent pharmacological strategy (PCSK9 inhibitors, higher intensity statins, or statins), whereas less intensive therapy corresponded to active control group or placebo/usual care of the trial. Metaregression and meta‐analyses were conducted using a random‐effects model. No significant association was noted between 1‐mmol/L reduction in LDL cholesterol and incident DM for more intensive lipid‐lowering therapy (risk ratio: 0.95; 95% CI, 0.87–1.04; P=0.30; R2=14%) or for statins or PCSK9 inhibitors. More intensive lipid‐lowering therapy was associated with a higher risk of incident DM compared with less intensive therapy (risk ratio: 1.07; 95% CI, 1.03–1.11; P\u3c0.001; I2=0%). These results were driven by higher risk of incident DM with statins (risk ratio: 1.10; 95% CI, 1.05–1.15; P\u3c0.001; I2=0%), whereas PCSK9 inhibitors were not associated with incident DM (risk ratio: 1.00; 95% CI, 0.93–1.07; P=0.96; I2=0%; P=0.02 for interaction). Conclusions Among intensive lipid‐lowering therapies, there was no independent association between reduction in LDL cholesterol and incident DM. The risk of incident DM was higher with statins, whereas PCSK9 inhibitors had no association with risk of incident DM

Association of Lowering Low�Density Lipoprotein Cholesterol With Contemporary Lipid�Lowering Therapies and Risk of Diabetes Mellitus: A Systematic Review and Meta�Analysis

Background The relationship between lowering LDL (low�density lipoprotein) cholesterol with contemporary lipid�lowering therapies and incident diabetes mellitus (DM) remains uncertain. Methods and Results Thirty�three randomized controlled trials (21 of statins, 12 of PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitors, and 0 of ezetimibe) were selected using Medline, Embase, and the Cochrane Central Register of Controlled Trials (inception through November 15, 2018). A total of 163 688 nondiabetic patients were randomly assigned to more intensive (83 123 patients) or less intensive (80 565 patients) lipid�lowering therapy. More intensive lipid�lowering therapy was defined as the more potent pharmacological strategy (PCSK9 inhibitors, higher intensity statins, or statins), whereas less intensive therapy corresponded to active control group or placebo/usual care of the trial. Metaregression and meta�analyses were conducted using a random�effects model. No significant association was noted between 1�mmol/L reduction in LDL cholesterol and incident DM for more intensive lipid�lowering therapy (risk ratio: 0.95; 95% CI, 0.87–1.04; P=0.30; R2=14%) or for statins or PCSK9 inhibitors. More intensive lipid�lowering therapy was associated with a higher risk of incident DM compared with less intensive therapy (risk ratio: 1.07; 95% CI, 1.03–1.11; P<0.001; I2=0%). These results were driven by higher risk of incident DM with statins (risk ratio: 1.10; 95% CI, 1.05–1.15; P<0.001; I2=0%), whereas PCSK9 inhibitors were not associated with incident DM (risk ratio: 1.00; 95% CI, 0.93–1.07; P=0.96; I2=0%; P=0.02 for interaction). Conclusions Among intensive lipid�lowering therapies, there was no independent association between reduction in LDL cholesterol and incident DM. The risk of incident DM was higher with statins, whereas PCSK9 inhibitors had no association with risk of incident DM