Mark J. Novak

Antimicrobial activity of tryptanthrins in Escherichia coli.

Tryptanthrins have potential therapeutic activity against a wide variety of pathogenic organisms, although little is known about their mechanism. Activity against Escherichia coli, however, has not been examined. The effects of tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) and nine derivatives on growth, survival, and mutagenesis in E. coli were examined. Analogues with a nitrogen atom at the 4-position of tryptanthrin stopped log phase growth of E. coli cultures at concentrations as low as 5 microM. Tryptanthrins decreased viability during incubation with cells in buffer by factors of 10(-2) to <10(-6) at 0.2-40 microM. Derivatives with an oxime group at the 6-position exhibited the greatest bactericidal activity. Most tryptanthrins were not mutagenic in several independent assays, although the 4-aza and 4 aza-8-fluoro derivatives increased frameshift mutations about 22- and 4-fold, respectively. Given the structure of trypanthrins, binding to DNA may occur by intercalation. From analysis using a sensitive linking number assay, several tryptanthrins, especially the 4-aza and 6-oximo derivatives, intercalate into DNA.

Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas) with pyridine nucleotide-driven flavoprotein electron transport systems

The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electron acceptor, various sulfur or nitrogen mustards and the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a(N)=15.483 G and a(H)=2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.

Novel computational study on π-stacking to understand mechanistic interactions of Tryptanthrin analogues with DNA

Abstract Based on recently published initial experimental results on the intercalation of a class of broad spectrum antiparasitic compounds, we present a purely theoretical approach for determining if these compounds may preferentially intercalate with guanosine/cytosine (GC)-rich or adenosine/thymidine (TA)-rich regions of DNA. The predictive model presented herein is based upon utilization of density functional theory (DFT) to determine a priori how the best intercalator may energetically and sterically interact with each of the nucleoside base pairs. A potential new method using electrostatic potential maps (EPMs) to visually select the best poses is introduced and compared to the existing brute-force center of mass (COM) approach. The EPM and COM predictions are in agreement with each other, but the EPM method is potentially much more efficient. We report that 4-azatryptantrin, the best intercalator, is predicted to favor π-stacking with GC over that of TA by approximately 2–4 kcal/mol. This represents a significant difference if one takes into account the Boltzmann distribution at physiological temperature. This theoretical method will be utilized to guide future experimental studies on the elucidation of possible mechanism(s) for the action of these antiparasitic compounds at the molecular level.

Effect of Adrenalin, Adrenochrome, and Adrenolutin on Connexin Proteins in the Cardiovasculature

Reported myocardial pathology resulting from increased levels of catecholamines in vivo has led us to investigate the effect of adrenalin on the gap junction proteins connexin 40 (Cx40) and Cx43 and the possible relationship to vascular toxicity. Adrenalin and its known metabolites, adrenochrome and adrenolutin, were used in this study. Utilizing the A7r5 rat aortic cell line, we evaluated the effects of adrenalin, adrenochrome, and adrenolutin on the expression and function of connexin 40 and 43 that are present in both cardiac and vascular tissues.

In silico simulations of tunneling barrier measurements for molecular orbital-mediated junctions: A molecular orbital theory approach to scanning tunneling microscopy

A new simulator for scanning tunneling microscopy (STM) is presented based on the linear combination of atomic orbitals molecular orbital (LCAO-MO) approximation for the effective tunneling Hamiltonian, which leads to the convolution integral when applied to the tip interaction with the sample. This approach intrinsically includes the structure of the STM tip. Through this mechanical emulation and the tip-inclusive convolution model, dI/dz images for molecular orbitals (which are closely associated with apparent barrier height, ϕap) are reported for the first time. For molecular adsorbates whose experimental topographic images correspond well to isolated-molecule quantum chemistry calculations, the simulator makes accurate predictions, as illustrated by various cases. Distortions in these images due to the tip are shown to be in accord with those observed experimentally and predicted by other ab initio considerations of tip structure. Simulations of the tunneling current dI/dz images are in strong agreeme...

NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for elucidating the metabolism of xenobiotics, as it allows for the least ambiguous assignment of chemical structure when compared to other forms of spectroscopy. In addition, it is a sensitive technique that can reveal the presence of transient species that otherwise would not be detected by utilizing either large‐scale batch processes or other forms of spectroscopic analyses. The primary focus of this unit describes the use of NMR to identify metabolites arising from the oxidation of thiodiglycol by equine and human variants of alcohol dehydrogenase (ADH). Given that it is often risky to base metabolism studies on a single form of spectroscopy, a spectrophotometric method is also presented. In addition, incorporation of independent organic syntheses in conjunction with the spectroscopic studies to further solidify structural identification of the ADH metabolites is presented.