QM/MM Analysis of Transition States and Transition State Analogues in Metalloenzymes.

Enzymology is approaching an era where many problems can benefit from computational studies. While ample challenges remain in quantitatively predicting behavior for many enzyme systems, the insights that often come from computations are an important asset for the enzymology community. Here we provide a primer for enzymologists on the types of calculations that are most useful for mechanistic problems in enzymology. In particular, we emphasize the integration of models that range from small active-site motifs to fully solvated enzyme systems for cross-validation and dissection of specific contributions from the enzyme environment. We then use a case study of the enzyme alkaline phosphatase to illustrate specific application of the methods. The case study involves examination of the binding modes of putative transition state analogues (tungstate and vanadate) to the enzyme. The computations predict covalent binding of these ions to the enzymatic nucleophile and that they adopt the trigonal bipyramidal geometry of the expected transition state. By comparing these structures with transition states found through free energy simulations, we assess the degree to which the transition state analogues mimic the true transition states. Technical issues worth treating with care as well as several remaining challenges to quantitative analysis of metalloenzymes are also highlighted during the discussion.

An intriguing association between ancestral mortality and male affective disorder.

We examine the relationship between ancestral age at death and affective disorder. A cohort of 204 socioeconomically favored men was selected for mental and physical health 50 years ago and followed up until age 69 +/- 1 (+/- SD) years in an interdisciplinary study. During follow-up, 49 of these men were identified at some point as being psychosocially impaired. Of these 49 men, 25 were identified as having probable affective disorder based on family history of affective illness, clinical signs, subjective symptoms, clinical diagnosis, and choice of pharmacological treatment. Objective signs and symptoms distinguished these 25 men from the remaining 24 men, whose adjustment over the 50 years was equally psychosocially impaired, but who were never noted to be significantly depressed. The mean age at death of the maternal grandfathers for the 25 depressed men was 60.4 years, significantly younger than the mean age at death of maternal grandfathers for either the total sample (70.1 years) or the 25 psychosocially impaired but not depressed men (68.8 years). The mean age at death of the depressed men's other five first-degree ancestors was not significantly different from the age at death of the ancestors of the rest of the sample. If depressed alcohol abusers were excluded, the mean age at death of maternal grandfathers of alcohol abusers did not differ from that of the controls' maternal grandfathers. The evidence for possible X-chromosome linkage in male psychobiological vulnerability to affective spectrum disorder is discussed.

Derivatization procedures for detection of prostaglandins in biological matrices by liquid chromatography/electrochemistry.

Conventional reversed-phase HPLC conditions have been optimized for resolution of a mixture containing prostaglandins PGE(1), PGE(2), PGF(1alpha), and PGF(2alpha). Electroactive derivative-forming reagents, such as p-nitrobenzyloxyamine, 2-bromo-2'-nitroacetophenone, and 2,4-dinitrophenylhydrazine have been evaluated for use as precolumn reagents for forming prostaglandin derivatives. The results indicate that detection limits of 120 pg are achievable with amperometric detection. The utility of the procedures developed is illustrated by the detection of prostaglandins in human urine and plasma.

Supercritical fluid extraction method development for extraction of an experimental HIV protease inhibitor drug from animal feed.

Supercritical fluid extraction (SFE) was evaluated as a sample preparation procedure for the recovery of experimental drugs from animal feed preparations that are generated during long-term toxicology studies. A commercially available supercritical fluid extractor was utilized to develop and validate an off-line procedure for the recovery of an experimental HIV protease inhibitor drug from animal feed. Extracts were analyzed with a conventional reversed-phase HPLC method. Elements of the SFE method developed that are described include optimization of the system temperature and selection of the extraction media modifier. The study emphasized the performance of two-day precision and accuracy studies. Precision and accuracy studies were carried out with SC-52151 levels of 0.05, 0.1 and 1.0% (w/w) and used an internal standard quantitation format. Also, the study utilized a relatively large analytical scale extraction vessel size of 10 ml to accomodate 6 g animal feed samples.

Comparison of drug substance impurity profiles generated with extended length columns during packed-column SFC.

The current study assesses the effect of extending column length during gradient packed column sub/supercritical fluid chromatography (PCSFC) experiments on the detection of known and unknown impurities in a drug substance sample. Quantitative drug substance impurity profiles were generated and compared using multiple column PCSFC and HPLC conditions. Also, chromatographic figures of merit were estimated and compared for components of a standard mixture during PCSFC experiments, which used one column, four columns, and six columns in series.

Characterization of polymorphs of a new anti-inflammatory drug.

The present study demonstrates the utility of a diversified analytical approach for the characterization and quantitative analysis for two polymorphs of a new anti-inflammatory agent, (+/-)-7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy-3,4-dihydro -8-propyl- 2H-1-benzopyran-2-carboxylic acid (SC-41930). The existence of two distinct crystal polymorphs of SC-41930 was qualitatively indicated through microscopy and application of thermal methods of analysis. The application of TGA was important for establishing that the two solid forms were, in fact, polymorphs, as opposed to solvated and unsolvated drug substances. The application of IR spectrometry revealed spectral features in the carbonyl stretching region, which were characteristic and unique to the two SC-41930 polymorphs. DRIFT spectrometry was implemented as the sampling method of choice to eliminate the possibility of polymorphic transformations during conventional mulling or KBr pellet sampling procedures. The DRIFT spectrometry procedure permitted development of a quantitative assay for detection of the low-melting polymorph (as an impurity) in high-melting samples. Calibration plots showed acceptable linearity of response from 0 to 25% (w/w) low-melting samples spiked into the high-melting polymorph. The performance characteristics of the method indicated good run-to-run and day-to-day consistency for its intended use.

Demonstrative validation study employing a packed column pressurized fluid chromatography method that provides assay, achiral impurities, chiral impurity, and IR identity testing for a drug substance.

Assay development, assay validation, and documentation are reported here for a single packed column pressurized fluid chromatographic/ultraviolet (UV) method that provides: (1) simultaneous detection and quantification for the chiral drug, the chiral impurity and seven achiral impurities; and (2) a Fourier transform infrared (FT-IR) spectrometric identification test result for the Searle drug substance sample, xemilofiban. The separation is achieved in less than 30 min with three columns in tandem and a gradient of CO2-CH3OH. The post-column flow is split between UV (assay) and FT-IR (identification). Precision and accuracy are consistent within figures of merit obtained by liquid chromatographic-ultraviolet assays on analogous drug substances. The reported procedure combines three typical drug substance tests into one test (e.g. chiral impurities, achiral impurities, and infrared identification).

Supercritical fluid extraction-liquid chromatography method development for a polymeric controlled-release drug formulation.

We have recently been involved in the development of a method for assaying the active component in a controlled-release drug formulation, which is composed of a drug substance covalently bonded to polymer matrix. The drug substance in the formulation is the active enantiomer of misoprostol, a synthetic analog of natural prostaglandins and the active ingredient in Cytotec. Our method development consisted of a systematic evaluation of dynamic, off-line supercritical fluid extraction (SFE) as sample preparation for the formulation assay. Extracts were analyzed with normal phase and reversed-phase HPLC methods. The reversed-phase system utilized postcolumn reaction to provide selective detection of the extracted prostaglandin sample components. Several SFE parameters were investigated to optimize the recovery of the drug substance from the formulation, including sample quantity, extraction cell volume, extraction duration, supercritical carbon dioxide modifier, temperature, pressure, and collection solvent. The SFE experiments were completed with a commercially available multicell extractor. Preliminary validation studies utilized a formulation made with radiolabeled drug to determine the recovery achieved under the optimized SFE conditions and assessed the precision of replicate determinations. Analysis was completed under the optimized conditions to quantitate levels of the active component and related compounds in lots of the experimental polymeric formulation and to determine the total weight per cent extracted.

Determination of hydroxylated aromatic compounds produced via superoxide-dependent formation of hydroxyl radicals by liquid chromatography/electrochemistry.

Hydroxyl radicals may be formed in a xanthine oxidase/hypoxanthine system, where the superoxide anion radical O-.2 and H2O2 are produced. The superoxide-dependent production of the OH. radicals may be monitored by determining the amount of hydroxylated aromatic compounds formed in such a system. Liquid chromatography/electrochemistry is a powerful tool for the determination of hydroxylated aromatic compounds. A technique is presented in which aniline and phenol are hydroxylated in xanthine oxidase/hypoxanthine incubations. No sample derivatization is needed for the determinations which can be accomplished by direct injection of the incubation mixture. Detection limits for 1,2- and 1,4-hydroxylated compounds are in the picomole range.

1995 IPA/Bayer Research Awards in Psychogeriatrics. Late-life consequences of affective spectrum disorder.

Recent research suggests that affective disorder is associated with increased mortality and physical morbidity, but the reasons for this association remain uncertain. This report describes a 50-year prospective study of 240 men evaluated from the time they were university students in 1940-1942. A family history of mental illness was obtained and the men's habits, psychological adjustment, and marital and occupational satisfaction were followed every 2 years and their objective physical health was tracked every 5 years until age 70. Twenty-five men were identified as having affective spectrum disorder prior to age 53. Of the variables studied, the presence of affective spectrum disorder was the most powerful predictor of poor psychosocial outcome at age 65 and one of the most powerful predictors of poor physical health. Alcohol abuse and cigarette abuse accounted for the observed increased rates of heart disease and cancer. When alcohol abuse, smoking, and suicide were controlled for, affective disorder made a significant contribution to physical morbidity by age 70, but not to mortality from natural causes. Affective spectrum disorder, even in an educated population without antisocial trends, carries a profound negative risk to late-life physical and social adjustment.