Discovery and characterization of small molecule inhibitors of the aldehyde dehydrogenase 1/2 family

Indiana University-Purdue University Indianapolis (IUPUI)The human aldehyde dehydrogenase (ALDH) superfamily consists of 19 isoenzymes that are critical for normal physiology as well as the removal of toxic aldehydes. Members of the ALDH1/2 family have vital roles in cell signaling during early development, ethanol metabolism, and the removal of aldehydes derived from oxidative stress. We sought to develop selective compounds toward ALDH2 to help determine its individual contribution to biological function, as many of the ALDH1/2 family possess overlapping substrate preferences. A high-throughput screen of over 100,000 compounds uncovered a class of aromatic lactones which inhibit the ALDH1/2 enzyme family. The lactones were then characterized using a combination of enzyme kinetics, X-ray crystallography, and cell culture experiments. We found that many of the lactones are over ten times more potent toward ALDH2 than daidzin, a previously described ALDH2 inhibitor. Our ability to produce many more ALDH isoenzymes allowed us to determine that daidzin is not as selective as previously believed, inhibiting ALDH2, ALDH1B1, and ALDH1A2 with equal potency. This inhibition pattern was seen with several of the aromatic lactones as well. Structural studies show that many of the lactones bind between key aromatic residues in the ALDH1/2 enzyme substrate-binding sites. One lactone in particular mimics the position of an aldehyde substrate and alters the position of the catalytic cysteine to interfere with the productive binding of NAD+ for enzyme catalysis. Further characterization of related compounds led to the realization that the mechanism of inhibition, potency, and selectivity differs amongst the lactones based off the substituents on the aromatic scaffold and its precise binding location. Two of these compounds were found to be selective for one of the ALDH1/2 family members, BUC22, selective for ALDH1A1, and BUC27, selective for ALDH2. BUC22 demonstrates ten-fold selectivity for ALDH1A1 over ALDH1A2 and does not inhibit the remaining ALDH1/2 enzymes. Additionally, treatment with BUC22 led to decreased growth of triple-negative breast cancer cells in culture. BUC27 inhibits ALDH2 with the same potency as daidzin. Both BUC22 and BUC27 could be further developed to use as chemical tools to better understand the functional roles of ALDH1A1 and ALDH2 in biological systems

LANDSAT inventory of surface-mined areas using extendible digital techniques

Multispectral analysis of LANDSAT imagery provides a rapid and accurate means of identification, classification, and measurement of strip-mined surfaces in Western Maryland. Four band analysis allows distinction of a variety of strip-mine associated classes, but has limited extendibility. A method for surface area measurement of strip mines, which is both geographically and temporally extendible, was developed using band-ratioed LANDSAT reflectance data. The accuracy of area measurement by this method, averaged over three LANDSAT scenes taken between September 1972 and July 1974, is greater than 93%. Total affected acreage of large (50 hectare/120 acre) mines can be measured to within 1.0%

Satellite data for surface-mine inventory

To determine the feasibility of satellite data for surface-mine inventory, particularly as it applies to coal, a case study was conducted in Maryland. A band-ratio method was developed to measure disturbed surface areas, and it proved to be extendible both temporally and geographically. This method was used to measure area changes in the region over three time periods from September 1972 through July 1974 and to map the entire two-county area for 1973. For mines ranging between 31 and 244 acres (12 to 98 hectares) the measurement accuracy of total affected acreage was determined to be 92%. Mines of 120 acres (50 hectares) and larger were measured with greater accuracy, some within one percent of the actual area. The ability to identify, classify, and measure strip-mine surfaces in a two-county area (1,541 square kilometers - 595 square miles) of western Maryland was demonstrated through the use of computer processing. On the basis of these results the use of LANDSAT satellite data and multilevel sampling of aircraft and field verification inspections, multispectral analysis of digital data is shown to be an effective, rapid, and accurate means of monitoring the surface mining cycle

Inhibition of the Aldehyde Dehydrogenase 1/2 Family by Psoralen and Coumarin Derivatives

Aldehyde dehydrogenase 2 (ALDH2), one of 19 ALDH superfamily members, catalyzes the NAD+-dependent oxidation of aldehydes to their respective carboxylic acids. In this study, we further characterized the inhibition of four psoralen and coumarin derivatives toward ALDH2 and compared them to the ALDH2 inhibitor daidzin for selectivity against five ALDH1/2 isoenzymes. Compound 2 (Ki = 19 nM) binds within the aldehyde-binding site of the free enzyme species of ALDH2. Thirty-three structural analogs were examined to develop a stronger SAR profile. Seven compounds maintained or improved upon the selectivity toward one of the five ALDH1/2 isoenzymes, including compound 36, a selective inhibitor for ALDH2 (Ki = 2.4 μM), and compound 32, which was 10-fold selective for ALDH1A1 (Ki = 1.2 μM) versus ALDH1A2. Further medicinal chemistry on the compounds’ basic scaffold could enhance the potency and selectivity for ALDH1A1 or ALDH2 and generate chemical probes to examine the unique and overlapping functions of the ALDH1/2 isoenzymes

Discovery of a series of aromatic lactones as ALDH1/2-directed inhibitors

In humans, the aldehyde dehydrogenase superfamily consists of 19 isoenzymes which mostly catalyze the NAD(P)(+)-dependent oxidation of aldehydes. Many of these isoenzymes have overlapping substrate specificities and therefore their potential physiological functions may overlap. Thus the development of new isoenzyme-selective probes would be able to better delineate the function of a single isoenzyme and its individual contribution to the metabolism of a particular substrate. This specific study was designed to find a novel modulator of ALDH2, a mitochondrial ALDH isoenzyme most well-known for its role in acetaldehyde oxidation. 53 compounds were initially identified to modulate the activity of ALDH2 by a high-throughput esterase screen from a library of 63,000 compounds. Of these initial 53 compounds, 12 were found to also modulate the oxidation of propionaldehyde by ALDH2. Single concentration measurements at 10μM compound were performed using ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and/or ALDH5A1 to determine the selectivity of these 12 compounds toward ALDH2. Four of the twelve compounds shared an aromatic lactone structure and were found to be potent inhibitors of the ALDH1/2 isoenzymes, but have no inhibitory effect on ALDH3A1, ALDH4A1 or ALDH5A1. Two of the aromatic lactones show selectivity within the ALDH1/2 class, and one appears to be selective for ALDH2 compared to all other isoenzymes tested

Charge neutralization in vacuum for non-conducting and isolated objects using directed low-energy electron and ion beams

We propose using ions and electrons of energy 1 eV–10 eV for neutralizing the charges on the non-conducting or isolated surfaces of high-sensitivity experiments. The mirror surfaces of the test masses of the laser interferometer gravitational observatory are used as an example of the implementation of this method. By alternatively directing beams of positive and negative charges towards the mirror surfaces, we ensure the neutralization of the total charge as well as the equalization of the surface charge distribution to within a few eV of the potential of the ground reference of the vacuum system. This method is compatible with operation in high vacuum, does not require measuring the potential of the mirrors and is expected not to damage sensitive optical surfaces

Home births in the Mosvold health ward of KwaZulu

A community survey was carried out to determine the frequency and the methods of home deliveries in the Mosvold health ward in northern KwaZulu. Of a sample of 210 mothers interviewed 46% had given birth at home, and of these 48% were delivered by traditional birth attendants; 84% gave birth in a kneeling or sitting position. In 32% of cases handling of the umbilical stump was unhygienic and potentially tetanogenic. Asked their reason for giving birth at home, most mothers gave transport problems and' sudden or unexpected onset of labour as their main reason, although a majority of grand multiparas expressed a preference for home delivery. Various recommendations are made on the basis of these findings

The NINJA-2 catalog of hybrid post-Newtonian/numerical-relativity waveforms for non-precessing black-hole binaries

The Numerical INJection Analysis (NINJA) project is a collaborative effort between members of the numerical relativity and gravitational wave data analysis communities. The purpose of NINJA is to study the sensitivity of existing gravitational-wave search and parameter-estimation algorithms using numerically generated waveforms, and to foster closer collaboration between the numerical relativity and data analysis communities. The first NINJA project used only a small number of injections of short numerical-relativity waveforms, which limited its ability to draw quantitative conclusions. The goal of the NINJA-2 project is to overcome these limitations with long post-Newtonian - numerical relativity hybrid waveforms, large numbers of injections, and the use of real detector data. We report on the submission requirements for the NINJA-2 project and the construction of the waveform catalog. Eight numerical relativity groups have contributed 63 hybrid waveforms consisting of a numerical portion modelling the late inspiral, merger, and ringdown stitched to a post-Newtonian portion modelling the early inspiral. We summarize the techniques used by each group in constructing their submissions. We also report on the procedures used to validate these submissions, including examination in the time and frequency domains and comparisons of waveforms from different groups against each other. These procedures have so far considered only the $(ell,m)=(2,2)$ mode. Based on these studies we judge that the hybrid waveforms are suitable for NINJA-2 studies. We note some of the plans for these investigations

Implementation of higher-order absorbing boundary conditions for the Einstein equations

We present an implementation of absorbing boundary conditions for the Einstein equations based on the recent work of Buchman and Sarbach. In this paper, we assume that spacetime may be linearized about Minkowski space close to the outer boundary, which is taken to be a coordinate sphere. We reformulate the boundary conditions as conditions on the gauge-invariant Regge-Wheeler-Zerilli scalars. Higher-order radial derivatives are eliminated by rewriting the boundary conditions as a system of ODEs for a set of auxiliary variables intrinsic to the boundary. From these we construct boundary data for a set of well-posed constraint-preserving boundary conditions for the Einstein equations in a first-order generalized harmonic formulation. This construction has direct applications to outer boundary conditions in simulations of isolated systems (e.g., binary black holes) as well as to the problem of Cauchy-perturbative matching. As a test problem for our numerical implementation, we consider linearized multipolar gravitational waves in TT gauge, with angular momentum numbers l=2 (Teukolsky waves), 3 and 4. We demonstrate that the perfectly absorbing boundary condition B_L of order L=l yields no spurious reflections to linear order in perturbation theory. This is in contrast to the lower-order absorbing boundary conditions B_L with L<l, which include the widely used freezing-Psi_0 boundary condition that imposes the vanishing of the Newman-Penrose scalar Psi_0.Comment: 25 pages, 9 figures. Minor clarifications. Final version to appear in Class. Quantum Grav

N,N-diethylaminobenzaldehyde (DEAB) as a substrate and mechanism-based inhibitor for human ALDH isoenzymes

N,N-diethylaminobenzaldehyde (DEAB) is a commonly used "selective" inhibitor of aldehyde dehydrogenase isoenzymes in cancer stem cell biology due to its inclusion as a negative control compound in the widely utilized Aldefluor assay. Recent evidence has accumulated that DEAB is not a selective inhibitory agent when assayed in vitro versus ALDH1, ALDH2 and ALDH3 family members. We sought to determine the selectivity of DEAB toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH3A1, ALDH4A1 and ALDH5A1 isoenzymes and determine the mechanism by which DEAB exerts its inhibitory action. We found that DEAB is an excellent substrate for ALDH3A1, exhibiting a Vmax/KM that exceeds that of its commonly used substrate, benzaldehyde. DEAB is also a substrate for ALDH1A1, albeit an exceptionally slow one (turnover rate ∼0.03 min(-1)). In contrast, little if any turnover of DEAB was observed when incubated with ALDH1A2, ALDH1A3, ALDH1B1, ALDH2 or ALDH5A1. DEAB was neither a substrate nor an inhibitor for ALDH1L1 or ALDH4A1. Analysis by enzyme kinetics and QTOF mass spectrometry demonstrates that DEAB is an irreversible inhibitor of ALDH1A2 and ALDH2 with apparent bimolecular rate constants of 2900 and 86,000 M(-1) s(-1), respectively. The mechanism of inactivation is consistent with the formation of quinoid-like resonance state following hydride transfer that is stabilized by local structural features that exist in several of the ALDH isoenzymes