Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

Background

α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.

Methodology/Principal Findings

Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.

Conclusions/Significance

We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD12 inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD12 selective inhibitors.     

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.     

Pleiotropic IFN-dependent and -independent effects of IRF5 on the pathogenesis of experimental lupus

Genetic polymorphisms of IFN regulatory factor 5 (IRF5) are associated with an increased risk of lupus in humans. In this study, we examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF5(-/-) mice shared many features with type I IFN receptor (IFNAR)(-/-) and TLR7(-/-) mice: production of anti-Sm/RNP autoantibodies, glomerulonephritis, generation of Ly6C(hi) monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation following pristane injection was greatly diminished in IRF5(-/-) mice, and Th cell differentiation was deviated from Th1 in wild-type mice toward Th2 in IRF5(-/-) mice. Th cell development was skewed similarly in TLR7(-/-) or IFNAR(-/-) mice, suggesting that IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not recruited to the site of inflammation in IRF5(-/-) or MyD88(-/-) mice, but were recruited normally in IFNAR(-/-) and TLR7(-/-) mice. In striking contrast to wild-type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF5(-/-) mice, suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data indicate that altered production of IFN-I and other cytokines in IRF5(-/-) mice prevents pristane from inducing lupus pathology by broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I-independent role of IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets.     

Systems biology of yeast cell death

Programmed cell death (PCD) (including apoptosis) is an essential process, and many human diseases of high prevalence such as neurodegenerative diseases and cancer are associated with deregulations in the cell death pathways. Yeast Saccharomyces cerevisiae, a unicellular eukaryotic organism, shares with multicellular organisms (including humans) key components and regulators of the PCD machinery. In this article, we review the current state of knowledge about cell death networks, including the modeling approaches and experimental strategies commonly used to study yeast cell death. We argue that the systems biology approach will bring valuable contributions to our understanding of regulations and mechanisms of the complex cell death pathways.     

Posters. P13 Cytohistological correlation in 212 FNAs of thyroid: analysis of discordant cases

Aim To detect major pitafalls in thyroid FNA and to confirm its in a clinical sensitivity and specificity. Methods A total of 9251 fine needle aspirations biopsy carried out at Bellaria Hospital in Bologna from 1991 to 2000 by a pathologist in the FNA Clinic or by a clinician under ultrasonic guidance using a small needle (25–27 G); at least two passes have been made for each nodule. The specimen was considered satisfactory if at least five groups of follicular cells with at least 10 cells each, were seen. The cytological results were tiered in a four categories classification: inadequate, negative, suspicious and positive. Cyto‐histological correlations were available in 212 cases: 127 benign lesions and 85 malignant lesions. An analysis of false positive cases and false negative cases was performed and discordant case reviewed according to the flowing criteria: architecture, cellularity, colloid, pseudoinclusions, nuclear groovings, chromatin pattern, nuclear membrane, cytoplasm, naked nuclei and lymphocytes. Results Diagnostic distribution in 9251 FNAs from the thyroid: 88.6% negatives, 2.8 suspicious, 2.4% positives and 6.2% inadequates. Specificity was 85.8% and sensitivity was 78.8%. Among the 18 false negative cases eight were papillary microcarcinomas, four papillary carcinomas, five follicular carcinoma and one a Hurtle cell carcinoma. Four false positive cases were found: three reported as papillary carcinomas and one as carcinoma NOS. Review of false positives showed that in three cases the colloid was fluid, in three cases nuclear grooving was rare or absent, in two cases degenerative vacuoles at MGG were interpreted as nuclear inclusions and in three cases benign naked nuclei were present in the background. Review of false negatives confirmed lack of malignant features in 13 (eight papillary microcarcinomas and five follicular carcinomas), five were interpretation errors (three papillary carcinomas, one follicular, one Hurtle cell). Conclusion FNAC of the thyroid is a sensitive and specific method of assessment for thyroid nodules but false negative and false positive cases do occur. Use of all and only few criteria enhances diagnostic accuracy.     

Signet-ring follicular adenoma of the thyroid diagnosed by fine needle aspiration. Report of a case with cytologic description

BACKGROUND: Follicular cells of the thyroid may undergo squamous, oncocytic or clear cell metaplastic changes. Of these, the clear cell change with signet-ring formation is the most unusual, and follicular adenoma of the thyroid with signet-ring formation is extremely rare. We describe for the first time the cytologic features of a clear cell adenoma of the thyroid obtained by fine needle aspiration (FNA). CASE: A 48-year-old woman in a euthyroid state presented with a 2-cm, right-sided thyroid nodule. Smears obtained by FNA of the nodule revealed clusters of large signet-ring cells in a bloody background. The signet-ring cells were round to oval, with large cytoplasmic vacuoles and hyperchromatic, eccentric nuclei. Colloid in the background was very scanty. Histologic examination of the right hemithyroidectomy specimen revealed a signet-ring follicular adenoma. CONCLUSION: Lack of familiarity with signet-ring cell adenoma of the thyroid could lead to an erroneous diagnosis of metastatic signet-ring cell carcinoma.     

CD24 and APC Genetic Polymorphisms in Pancreatic Cancers as Potential Biomarkers for Clinical Outcome

Background

There are no validated biomarkers that correlate with the prognosis of pancreatic ductal adenocarcinoma (PDA). The CD24 and adenomatous polyposis coli (APC) genes are important in the malignant transformation of gastrointestinal cells. This study examined APC and CD24 genetic polymorphisms and their possible impact on survival of patients with PDA.

Methods

Clinical and pathological data as well as blood samples for extracting DNA were obtained for 73 patients with PDA. Real-time PCR assessed genetic variants of APC (I1307K and E1317Q), and four different single nucleotide polymorphisms (SNPs) in the CD24 gene: C170T (rs52812045), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818).

Results

The median age at diagnosis was 64 (41–90) years. Thirty-one patients (42.5%) were operable, 16 (22%) had locally advanced disease and 26 (35.5%) had disseminated metastatic cancer. The malignancy-related mortality rate was 84%. Median survival was 14 months (11.25–16.74). Survival was similar for wild-type (WT), heterozygous and homozygous variants of the APC or CD24 genes. The three most frequent CD24 SNP combinations were: heterozygote for A1626G and WT for the rest of the alleles (14% of patients), heterozygote for C170T, A1626G, A1056G and WT for the rest (14% of patients), and heterozygote for C170T, A1056G and WT for the rest (10% of patients). All patients were APC WT. The first two groups were significantly younger at diagnosis than the third group.

Conclusions

Specific polymorphisms in the APC and CD24 genes may play a role in pancreatic cancer development. Correlation with survival requires a larger cohort.     

The newly synthesized pool of dopamine determines the severity of methamphetamine-induced neurotoxicity

The neurotransmitter dopamine (DA) has long been implicated as a participant in the neurotoxicity caused by methamphetamine (METH), yet, its mechanism of action in this regard is not fully understood. Treatment of mice with the tyrosine hydroxylase (TH) inhibitor α-methyl- p -tyrosine (AMPT) lowers striatal cytoplasmic DA content by 55% and completely protects against METH-induced damage to DA nerve terminals. Reserpine, by disrupting vesicle amine storage, depletes striatal DA by more than 95% and accentuates METH-induced neurotoxicity. l -DOPA reverses the protective effect of AMPT against METH and enhances neurotoxicity in animals with intact TH. Inhibition of MAO-A by clorgyline increases pre-synaptic DA content and enhances METH striatal neurotoxicity. In all conditions of altered pre-synaptic DA homeostasis, increases or decreases in METH neurotoxicity paralleled changes in striatal microglial activation. Mice treated with AMPT, l -DOPA, or clorgyline + METH developed hyperthermia to the same extent as animals treated with METH alone, whereas mice treated with reserpine + METH were hypothermic, suggesting that the effects of alterations in cytoplasmic DA on METH neurotoxicity were not strictly mediated by changes in core body temperature. Taken together, the present data reinforce the notion that METH-induced release of DA from the newly synthesized pool of transmitter into the extracellular space plays an essential role in drug-induced striatal neurotoxicity and microglial activation. Subtle alterations in intracellular DA content can lead to significant enhancement of METH neurotoxicity. Our results also suggest that reactants derived from METH-induced oxidation of released DA may serve as neuronal signals that lead to microglial activation early in the neurotoxic process associated with METH.     

Atomic force microscopy measurement of leukocyte-endothelial interaction

Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40-100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-alpha-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against beta1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-alphaVbeta3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50 approximately 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-beta1 and anti-alphaVbeta3 antibodies. In conclusion, these data demonstrate the role played by beta1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by alphaVbeta3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.     

Segregation of the yeast plasmid: similarities and contrasts with bacterial plasmid partitioning

The high copy yeast plasmid 2 microm circle, like the well-studied low copy bacterial plasmids, utilizes two partitioning proteins and a cis-acting 'centromere'-like sequence for its stable propagation. Functionally, though, the protein and DNA constituents of the two partitioning systems are quite distinct. Key events in the yeast and bacterial segregation pathways are plasmid organization, localization, replication, 'counting' of replicated molecules and their distribution to daughter cells. We suggest that the two systems facilitate these common logistical steps by adapting to the physical, biochemical, and mechanical contexts in which the host chromosomes segregate.