There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65–90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.

The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species, including humans; however, the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis, we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro, and on spermatogenesis and fertility in vivo, in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC50 of 0.3 μM. In vivo, the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid, severely impaired spermatogenesis, and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible, nonhormonal male contraceptive.

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Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in (R) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles.

Pharmacological suppression of RA synthesis via inhibition of ALDH1A1 may be a potential target for treatment of obesity.

We report on the synthesis, kinetics of proteolysis by trypsin, and morphological characterization of a novel lipidated decapeptide that spontaneously self-assembles in aqueous solutions into 0.5 μm diameter hollow tubules and helices that range in length from tens to hundreds of micrometers depending on formation conditions. We also report on an improved method for the tritioacetylation of peptides. Tight molecular packing of the peptide−amphiphile into a crystalline bilayer array forces tight packing between peptide headgroups, which was found to significantly protect the peptides from proteolysis by trypsin. Relief of this steric hindrance between peptide headgroups caused by solubilization of the bilayer into detergent micelles accelerated the rate of trypsin hydrolysis by 32,000-fold. Raman spectroscopy and circular dichroism spectropolarimetry were used to gain molecular-level insight into the difference between hydrolysis rates. Results obtained from these studies suggest that differences in molecular packing and conformation of the peptide headgroups in crystalline tubular and dispersed micellar phases determine the extent of proteolytic protection. Protection from proteolysis is considered a useful feature of lipidated peptide tubules for their potential use as a depot of bioactive peptides and other labile prodrugs at defined biological sites for sustained release.

BackgroundOne of the major challenges health care systems face in modern time is treating chronic disorders. In recent years, the increasing occurrence of multiple chronic disorders (MCC) in single individuals has compounded the complexity of health care. In 2008, it was estimated that worldwide as many as one quarter of the population between the ages of sixty five to sixty nine suffered from two or more chronic conditions and this prevalence rose with age. Clinical guidelines provide guidance for management of single disorders, but not for MCC. The aim of the present study was the study of the prevalence, distribution and impact of MCC in a large Israeli health system.MethodsWe performed a cross-sectional study of MCC in the Maccabi Healthcare System (MHS), Israel’s second largest healthcare service, providing care for approximately two million people. Data regarding chronic conditions was collected through electronic medical records and organizational records, as was demographic and socioeconomic data. Age and sex specific data were compared with previously published data from Scotland.ResultsTwo thirds of the population had two or more chronic disorders. This is significantly higher than previously published rates. A correlation between patient age and number of chronic disorders was found, as was a correlation between number of chronic disorders and low socioeconomic status, with the exception of children due to a high prevalence of learning disabilities, asthma, and visual disturbances.DiscussionMCC is very prevalent in the MHS population, increases with age, and except for children is more prevalent in lower socioeconomic classes, possibly due to the a combination of the structure of the Israeli universal insurance and requirements of the ministry of education for exemptions and benefits. A higher than previously reported prevalence of MCC may be due to the longtime use of use of integrated electronic medical records.ConclusionsTo effectively deal with MCC health care systems must devise strategies, including but not limited to, information technologies that enable shared teamwork based on clinical guidelines which address the problem of multiple, as opposed to single chronic disorders in patients.