Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase.

Organophosphorus (OP) nerve agents and pesticides present significant threats to civilian and military populations. OP compounds include the nefarious G and V chemical nerve agents, but more commonly, civilians are exposed to less toxic OP pesticides, resulting in the same negative toxicological effects and thousands of deaths on an annual basis. After decades of research, no new therapeutics have been realized since the mid-1900s. Upon phosphylation of the catalytic serine residue, a process known as inhibition, there is an accumulation of acetylcholine (ACh) in the brain synapses and neuromuscular junctions, leading to a cholinergic crisis and eventually death. Oxime nucleophiles can reactivate select OP-inhibited acetylcholinesterase (AChE). Yet, the fields of reactivation of AChE and butyrylcholinesterase encounter additional challenges as broad-spectrum reactivation of either enzyme is difficult. Additional problems include the ability to cross the blood brain barrier (BBB) and to provide therapy in the central nervous system. Yet another complication arises in a competitive reaction, known as aging, whereby OP-inhibited AChE is converted to an inactive form, which until very recently, had been impossible to reverse to an active, functional form. Evaluations of uncharged oximes and other neutral nucleophiles have been made. Non-oxime reactivators, such as aromatic general bases and Mannich bases, have been developed. The issue of aging, which generates an anionic phosphylated serine residue, has been historically recalcitrant to recovery by any therapeutic approach-that is, until earlier this year. Mannich bases not only serve as reactivators of OP-inhibited AChE, but this class of compounds can also recover activity from the aged form of AChE, a process referred to as resurrection. This review covers the modern efforts to address all of these issues and notes the complexities of therapeutic development along these different lines of research.

Early Successes in an Open Access, Provincially Funded Hepatitis C Treatment Program in Prince Edward Island.

INTRODUCTION: The availability of curative hepatitis C therapies has created an opportunity to improve treatment delivery and access. Local providers, government, industry, and community groups in Prince Edward Island developed an innovative province-wide care model. Our goal was to describe the first year of program implementation. MATERIAL AND METHODS: Using a communitybased prospective observational study design, all chronic hepatitis C referrals received from April 2015 to April 2016 were recorded in a database. Primary analysis assessed the time from referral to assessment/treatment, as well as the number of referrals, assessments, and treatment initiations. Secondary objectives included: (1) treatment effectiveness using intention-to-treat analysis; and (2) patient treatment experience assessed using demographics, adverse events, and medication adherence. RESULTS: During the study period 242 referrals were received, 123 patients were seen for intake assessments, and 93 initiated direct-acting antiviral therapy based on medical need. This is compared to 4 treatment initiations in the previous 2 years. The median time from assessment to treatment initiation was 3 weeks. Eighty-two of 84 (97.6%, 95% CI 91.7 - 99.7%) patients for whom outcome data were available achieved sustained virologic response at 12 weeks post-treatment; 1 was lost to follow-up and 1 died from an unrelated event. In the voluntary registry, 39.7% of patients reported missed treatment doses. CONCLUSION: In conclusion, results from the first 12 months of this multi-phase hepatitis C elimination strategy demonstrate improved access to treatment, and high rates of safe engagement and cure for patients living with chronic hepatitis C genotype 1 infections.

Early Successes in an Open Access, Provincially Funded Hepatitis C Treatment Program in Prince Edward Island.

INTRODUCTION: The availability of curative hepatitis C therapies has created an opportunity to improve delivery and access. Local providers, government, industry, and community groups in Prince Edward Island developed an innovative province-wide care model. Our goal was to describe the first year of program implementation. MATERIAL AND METHODS: Using a community based prospective observational study design, all chronic hepatitis C referrals received from April 2015 to April 2016 were recorded in a database. Primary analysis assessed the time from referral to assessment/treatment, as well as the number of referrals, assessments, and treatment initiations. Secondary objectives included: 1) Treatment effectiveness using intention-to-treat analysis; and 2) Patient treatment experience assessed using demographics, adverse events, and medication adherence. RESULTS: During the study period 242 referrals were received, 123 patients were seen for intake assessments, and 93 initiated direct-acting antiviral therapy based on medical need. This is compared to 4 treatment initiations in the previous 2 years. The median time from assessment to treatment initiation was 3 weeks. Eighty-two of 84 (97.6%, 95% CI 91.7 - 99.7%) patients for whom outcome data were available achieved sustained virologic response at 12 weeks post-treatment; 1 was lost to follow-up and 1 died from an unrelated event. In the voluntary registry, 39.7% of patients reported missed treatment doses. CONCLUSION: In conclusion, results from the first 12 months of this multi-phase hepatitis C elimination strategy demonstrate improved access to treatment, and high rates of safe engagement and cure for patients living with chronic hepatitis C genotype 1 infections.

Quantitative structure-activity relationship models of chemical transformations from matched pairs analyses.

The concepts of activity cliffs and matched molecular pairs (MMP) are recent paradigms for analysis of data sets to identify structural changes that may be used to modify the potency of lead molecules in drug discovery projects. Analysis of MMPs was recently demonstrated as a feasible technique for quantitative structure-activity relationship (QSAR) modeling of prospective compounds. Although within a small data set, the lack of matched pairs, and the lack of knowledge about specific chemical transformations limit prospective applications. Here we present an alternative technique that determines pairwise descriptors for each matched pair and then uses a QSAR model to estimate the activity change associated with a chemical transformation. The descriptors effectively group similar transformations and incorporate information about the transformation and its local environment. Use of a transformation QSAR model allows one to estimate the activity change for novel transformations and therefore returns predictions for a larger fraction of test set compounds. Application of the proposed methodology to four public data sets results in increased model performance over a benchmark random forest and direct application of chemical transformations using QSAR-by-matched molecular pairs analysis (QSAR-by-MMPA).

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

C2 hydroxyl group governs the difference in hydrolysis rates of methyl-α-D-glycero-D-guloseptanoside and methyl-ß-D-glycero-D-guloseptanoside.

A computational investigation into the hydrolysis of two methyl septanosides, methyl-α-D-glycero-D-guloseptanoside and methyl-ß-D-glycero-D-guloseptanoside was undertaken. These septanosides were chosen as model compounds for comparison to methyl pyranosides and allowed direct comparison of α versus ß hydrolysis rates for a specific septanoside isomer. Results suggest that hydrolysis takes place without proceeding through a transition state, an observation that was suggested in previous computational studies on exocyclic bond cleavage of carbohydrates. A conformational analysis of α- and ß-anomers 1 and 2 and their corresponding oxocarbenium 3, coupled with relaxed potential energy surface (PES) scans (M06-2X/6-311+G**, implicit methanol), indicated that hydrolysis of the α-anomer is favored by 1-2 kcal/mol over the ß-anomer, consistent with experiment. Model systems revealed that the lowest energy conformations of the septanoside ring system destabilize the ß-anomer by 2-3 kcal/mol relative to the α-anomer, and the addition of a single hydroxyl group at the C2-position on a minimal oxepane acetal can reproduce the PES for the septanoside 1. These results suggest that the C2 hydroxyl plays a unique role in the hydrolysis mechanism, destabilizing the septanoside via its proximity to the anomeric carbon and also through its interaction with the departing methanol from the α-anomer via hydrogen-bonding interactions.

Jujitsu kick to the abdomen: a case of blunt abdominal trauma resulting in hematochezia and transient ischemic colitis.

Blunt abdominal trauma is a common presentation to the emergency department. Ischemic colitis is a rare complication of this and its possible sequelae are important for an emergency physician to recognize. A 21-year-old man presented to the emergency department with abdominal pain and hourly episodes of bright red blood per rectum shortly after being kicked in the stomach at his jujitsu class. He had no significant medical history, and results of his systems review were otherwise unremarkable. On examination, he appeared well, with normal vital signs. He had mild lower abdominal tenderness, but there were no peritoneal signs present. There was blood on the digital rectal examination. His hemoglobin, platelet, and international normalized ratio levels were normal and his abdominal radiograph was unremarkable. The gastroenterology service was contacted because of the hematochezia and a flexible sigmoidoscopy was performed. The sigmoidoscopy showed erythema, ulceration, and edema of a segment in the left colon, consistent with ischemic colitis. This was later confirmed on biopsy. A computed tomography (CT) scan of the abdomen was conducted, which revealed left colonic inflammation consistent with colonic ischemia. There was no mesenteric vascular thrombosis or mesenteric hematoma found on CT. His hematochezia and abdominal pain subsided spontaneously, and he was discharged home. This case illustrates transient ischemic colitis as a potential presentation of blunt abdominal trauma, and emergency physicians should consider this uncommon diagnosis in the differential diagnosis of patients presenting after abdominal trauma.

Chemical synthesis of two series of nerve agent model compounds and their stereoselective interaction with human acetylcholinesterase and human butyrylcholinesterase.

Both G and V type nerve agents possess a center of chirality about phosphorus. The S(p) enantiomers are generally more potent inhibitors than their R(p) counterparts toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To develop model compounds with defined centers of chirality that mimic the target nerve agent structures, we synthesized both the S(p) and the R(p) stereoisomers of two series of G type nerve agent model compounds in enantiomerically enriched form. The two series of model compounds contained identical substituents on the phosphorus as the G type agents, except that thiomethyl (CH(3)-S-) and thiocholine [(CH(3))(3)NCH(2)CH(2)-S-] groups were used to replace the traditional nerve agent leaving groups (i.e., fluoro for GB, GF, and GD and cyano for GA). Inhibition kinetic studies of the thiomethyl- and thiocholine-substituted series of nerve agent model compounds revealed that the S(p) enantiomers of both series of compounds showed greater inhibition potency toward AChE and BChE. The level of stereoselectivity, as indicated by the ratio of the bimolecular inhibition rate constants between S(p) and R(p) enantiomers, was greatest for the GF model compounds in both series. The thiocholine analogues were much more potent than the corresponding thiomethyl analogues. With the exception of the GA model compounds, both series showed greater potency against AChE than BChE. The stereoselectivity (i.e., S(p) > R(p)), enzyme selectivity, and dynamic range of inhibition potency contributed from these two series of compounds suggest that the combined application of these model compounds will provide useful research tools for understanding interactions of nerve agents with cholinesterase and other enzymes involved in nerve agent and organophosphate pharmacology. The potential of and limitations for using these model compounds in the development of biological therapeutics against nerve agent toxicity are also discussed.

Hydrolysis of nerve agents by model nucleophiles: a computational study.

Density functional theory calculations were employed to study the reaction of five nerve agents with model nucleophiles, including EtX(-) and EtXH (X=O, S, Se) for serine, cysteine and selenocysteine, respectively. Calculations at the B3LYP/6-311++G(2d,p) level of theory predict an exothermic reaction between ethoxide and all of the nerve agents studied. As compared to EtO(-) as a nucleophile, these reactions become approximately 30 kcal/mol more endothermic for EtS(-), and by approximately 40 kcal/mol for EtSe(-). The equivalent reactions with the neutral nucleophiles (EtXH) were more endothermic. The effect of solvation on the reaction thermochemistry was determined using a polarizable continuum model simulating the dielectric constant of chloroform. While there was a large exothermic shift for reactions involving charged nucleophiles with solvation modeling, the corresponding shift was minimal for the reaction with neutral nucleophiles.

Study of para-Quinone Methide Precursors toward the Realkylation of Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) is an essential enzyme that can be targeted by organophosphorus (OP) compounds, including nerve agents. Following exposure to OPs, AChE becomes phosphylated (inhibited) and undergoes a subsequent aging process where the OP-AChE adduct is dealkylated. The aged AChE is unable to hydrolyze acetylcholine, resulting in accumulation of the neurotransmitter in the central nervous system (CNS) and elsewhere. Current therapeutics are only capable of reactivating inhibited AChE. There are no known therapeutic agents to reverse the aging process or treat aged AChE. Quinone methides (QMs) have been shown to alkylate phosphates under physiological conditions. In this study, a small library of novel quinone methide precursors (QMPs) has been synthesized and examined as potential alkylating agents against model nucleophiles, including a model phosphonate. Computational studies have been performed to evaluate the affinity of QMPs for the aged AChE active site, and preliminary testing with electric eel AChE has been performed.

Implications of Dynamic Occupancy, Binding Kinetics, and Channel Gating Kinetics for hERG Blocker Safety Assessment and Mitigation.

Blockade of the hERG potassium channel prolongs the ventricular action potential (AP) and QT interval, and triggers early after depolarizations (EADs) and torsade de pointes (TdP) arrhythmia. Opinions differ as to the causal relationship between hERG blockade and TdP, the relative weighting of other contributing factors, definitive metrics of preclinical proarrhythmicity, and the true safety margin in humans. Here, we have used in silico techniques to characterize the effects of channel gating and binding kinetics on hERG occupancy, and of blockade on the human ventricular AP. Gating effects differ for compounds that are sterically compatible with closed channels (becoming trapped in deactivated channels) versus those that are incompatible with the closed/closing state, and expelled during deactivation. Occupancies of trappable blockers build to equilibrium levels, whereas those of non-trappable blockers build and decay during each AP cycle. Occupancies of ~83% (non-trappable) versus ~63% (trappable) of open/inactive channels caused EADs in our AP simulations. Overall, we conclude that hERG occupancy at therapeutic exposure levels may be tolerated for nontrappable, but not trappable blockers capable of building to the proarrhythmic occupancy level. Furthermore, the widely used Redfern safety index may be biased toward trappable blockers, overestimating the exposure-IC50 separation in nontrappable cases.

Fluorinated compounds in medicinal chemistry: recent applications, synthetic advances and matched-pair analyses.

In recent years, several new fluorinated functional groups have been employed in medicinal chemistry. This review will highlight some recent developments in this area. We draw attention to useful synthetic advances for the installation of fluorine-containing groups. In addition, we examine the application of some fluorinated functional groups that have recently been gaining popularity in drug discovery. We use matched-pair analysis to assemble aggregate data on the impact on potency of one of these groups, pentafluorosulfanyl, as compared to trifluoromethyl. We further used matchedpair analysis to identify some interesting effects on in vitro ADME properties of replacing H by F on certain moieties.

Reaction profiles of the interaction between sarin and acetylcholinesterase and the S203C mutant: model nucleophiles and QM/MM potential energy surfaces.

The phosphonylation mechanism of AChE and the S203C mutation by sarin (GB) is evaluated using two reaction schemes: a small model nucleophile (ethoxide, CH(3)CH(2)O(-)) and quantum mechanical/molecular mechanical (QM/MM) simulations. Calculations utilizing small model nucleophiles indicate that the reaction barrier for addition to GB is the rate-limiting step for both ethoxide and ethyl thiolate (CH(3)CH(2)S(-)); moreover, the activation barrier for addition to the phosphorus center of GB by ethyl thiolate is significantly larger (13.2 kcal/mol) than for ethoxide (8.3 kcal/mol). The decomposition transition state for both nucleophiles was determined to be approximately 1 kcal/mol. QM/MM simulations for AChE suggest a similar reaction mechanism for phosphonylation of the catalytic S203; however, the relative energetics are altered significantly compared to the isolated system. QM/MM results indicate that formation of the penta-coordinate intermediate is the rate-limiting step in the enzymatic system, with an activation barrier of 3.6 kcal/mol. Hydrogen-bonding interactions between the fluoride leaving group of GB with Y124 in AChE are observed throughout the reaction profile. The S203C mutation alters the relative energetics of the reaction, increasing the energy barrier for formation of the penta-coordinate intermediate to a value of 4.5 kcal/mol; moreover, the penta-coordinate intermediate (as product) is stabilized by an additional 6 kcal/mol when compared to wild-type AChE.

Butyrylcholinesterase and G116H, G116S, G117H, G117N, E197Q and G117H/E197Q mutants: a molecular dynamics study.

Butyrylcholinesterase (BuChE) is a stoichiometric bioscavenger against organophosphorus (OP) nerve agent poisoning, and efforts to make BuChE variants that are catalytically active against a wide spectrum of nerve agents have been ongoing for the last decade. In order to understand the structural consequences for BuChE, we carried out extensive molecular dynamics (MD) simulations on wild-type BuChE (PDB ID: 1P0I) and several known and new variants of this enzyme, but without the presence of any ligand in the active site. The MD simulations on WT-BuChE identified two labile orientations for the catalytic serine, and also showed the likelihood of a backdoor. Upon changes at the G116 position, severe alterations around the active site region were identified. Simulations on both G117H and G117N variants showed the existence of a bound water molecule that is in close proximity to S198. Modeling of the E197Q mutant suggested that Q197 can be in two distinct orientations, one similar to the E202Q-AChE crystal structure and another in proximity to G439 and E441. The double mutant, G117H/E197Q, was found to have structural characteristics of both G117H and E197Q. In light of the computational results, previous experimental observations are discussed.

Computational Modeling of Human Paraoxonase 1: Preparation of Protein Models, Binding Studies, and Mechanistic Insights.

The enzyme human paraoxonase 1 (huPON1) has demonstrated significant potential for use as a bioscavenger for treatment of exposure to organophosphorus (OP) nerve agents. Herein we report the development of protein models for the human isoform derived from a crystal structure of a chimeric version of the protein (pdb ID: 1V04) and a homology model derived from the related enzyme diisopropylfluorophosphatase (pdb ID: 1XHR). From these structural models, binding modes for OP substrates are predicted, and these poses are found to orient substrates in proximity to residues known to modulate specificity of the enzyme. Predictions are made with regard to the role that residues play in altering substrate binding and turnover, in particular with regard to the stereoselectivity of the enzyme, and the known differences in activity related to a natural polymorphism in the enzyme. Potential mechanisms of action of the protein for catalytic hydrolysis of OP substrates are also evaluated in light of the proposed binding modes.