Design and synthesis of aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors with enhanced brain permeability

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aβ40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..     

Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.     

Increased effect of the ApoE gene on survival at advanced age in healthy and long‐lived Danes: two nationwide cohort studies

Studies of Nordic twins suggest an increased genetic influence on mortality with age. Contrary to this, the heterogeneity hypothesis predicts that the mortality of individuals carrying a ‘frail’ or ‘risky’ genotype in a population will approach that of noncarriers with age because of selection pressure. The ApoE ε4 allele is associated with an increased mortality risk, and its effect has been suggested to decrease with age. Here, we investigated the effect of ApoE ε4 allele on survival in a sample of the healthiest and long‐lived Danes. The study population comprised Danes born in 1905 and a replicate sample of the 1895 cohort. For the 1905 cohort, a total of 350 carriers and 1256 noncarriers of the ApoE ε4 allele were followed from 1998 until death or end of follow‐up. Cox regression models were used for the analysis. Of the 1606 persons with known ApoE ε4 status in 1998, 1546 had died at the end of the 10‐year follow‐up. Carriers of the ApoE ε4 allele had an increased mortality compared to noncarriers, and the influence of ApoE status on mortality increased in the age interval 92–103. For the covariates sex and independency status, the difference in relative risk of death between groups decreased with advancing age. Our findings of increasing influence of ApoE ε4 allele on mortality with age do not support previous findings of decreased influence ApoE ε4 allele on mortality with age, and alternative models such as the multifactorial threshold models should be considered for understanding the genetic effects on mortality at advanced age.     

Striptease on glass: validation of an improved stripping procedure for in situ microarrays

Microarrays have rapidly become an indispensable tool for gene analysis. Microarray experiments can be cost prohibitive, however, largely due to the price of the arrays themselves. Whilst different methods for stripping filter arrays on membranes have been established, only very few protocols are published for thermal and chemical stripping of microarrays on glass. Most of these protocols for stripping microarrays on glass were developed in combination with specific surface chemistry and different coatings for covalently immobilizing presynthesized DNA in a deposition process. We have developed a method for stripping commercial in situ microarrays using a multi-step procedure. We present a method that uses mild chemical degradation complemented by enzymatic treatment. We took advantage of the differences in biochemical properties of covalently linked DNA oligonucleotides on in situ synthesized microarrays and the antisense cRNA hybridization probes. The success of stripping protocols for microarrays on glass was critically dependent on the type of arrays, the nature of sample used for hybridization, as well as hybridization and washing conditions. The protocol employs alkali hydrolysis of the cRNA, several enzymatic degradation steps using RNAses and Proteinase K, combined with appropriate washing steps. Stripped arrays were rehybridized using the same protocols as for new microarrays. The stripping method was validated with microarrays from different suppliers and rehybridization of stripped in situ arrays yielded comparable results to hybridizations done on unused, new arrays with no significant loss in precision or accuracy. We show that stripping of commercial in situ arrays is feasible and that reuse of stripped arrays gave similar results compared to unused ones. This was true even for biological samples that show only slight differences in their expression profiles. Our analyses indicate that the stripping procedure does not significantly influence data quality derived from post-primary hybridizations. The method is robust, easy to perform, inexpensive, and results after reuse are of comparable accuracy to new arrays.     

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.     

Unrevealed structural requirements for auxin-like molecules by theoretical and experimental evidences

An computational-biostatistical approach, supported by ab initio optimizations of auxin-like molecules, was used to find biologically meaningful relationships between quantum chemical variables and fresh bioassay's data. It is proven that the auxin-like recognition requires different molecular assembling states. We suggest that the carboxyl group is not the determining factor in explaining the biological auxin-like conduct. The biological effects depends essentially on the chemical condition of the ring system. The aim to find active molecules (quantum objects) via statistical grouping-analysis of molecular quantum similarity measures was verified by bioactivity assays. Next, this approach led to the discovery of a non-carboxylated active auxin-like molecule (2,6-dibromo-phenol). This is the first publication on structure activity relationship of auxin-like molecules, which relies on highly standardized bioassays of different auxins screened in parallel as well as analysed by multi-dimensional scaling.     

Clonal expansion of CD8+ effector T cells in childhood tuberculosis

The role of CD8(+) T cells in human tuberculosis (TB) remains elusive. We analyzed the T cell repertoire and phenotype in 1) children with active TB (< or =4 years), 2) healthy latently Mycobacterium tuberculosis-infected children, and 3) noninfected age-matched (tuberculin skin test-negative) controls. Ex vivo phenotyping of T cell subpopulations by flow cytometry revealed a significant increase in the proportion of CD8(+)CD45RO(-)CD62L(-)CD28(-)CD27(-) effector T cells (T(EF)) in the peripheral blood of children with active TB (22.1 vs 9.5% in latently M. tuberculosis-infected children, vs 8.5% in tuberculin skin test-negative controls). Analyses of TCR variable beta-chains revealed markedly skewed repertoires in CD8(+) T(EF) and effector memory T cells. Expansions were restricted to single TCR variable beta-chains in individual donors indicating clonal growth. CDR3 spectratyping and DNA sequencing verified clonal expansion as the cause for CD8(+) effector T cell enrichment in individual TB patients. The most prominent enrichment of highly similar T(EF) clones (>70% of CD8(+) T(EF)) was found in two children with active severe TB. Therefore, clonal expansion of CD8(+) T(EF) occurs in childhood TB with potential impact on course and severity of disease.     

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.