A sensitive and drug tolerant assay for detecting anti-AAV9 antibodies using affinity capture elution.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Angiopoietin-like protein 4(E40K) and ANGPTL4/8 complex have reduced, temperature-dependent LPL-inhibitory activity compared to ANGPTL4.

We previously demonstrated that angiopoietin-like 8 (ANGPTL8) forms a localized complex with ANGPTL4 to reduce its lipoprotein lipase (LPL)-inhibitory activity and enable increased postprandial uptake of fatty acids (FA) into adipose tissue. Because prolonged cold exposure may increase adipose tissue FA uptake and decrease circulating triglycerides (TG) by reducing ANGPTL4 expression and inducing ANGPTL8 expression (and thus ANGPTL4/8 expression), we investigated the effect of temperature on ANGPTL4 and ANGPTL4/8 LPL-inhibitory activities in vitro. As the ANGPTL4(E40K) mutation results in decreased TG, we also characterized ANGPTL4(E40K) and ANGPTL4(E40K)/8 complex LPL-inhibitory activities. Interestingly, while ANGPTL3, ANGPTL3/8, and ANGPTL4 showed similar LPL inhibition at 37 °C and 22 °C, the already reduced LPL-inhibitory activity of ANGPTL4/8 at 37 °C was even more decreased at 22 °C. At 37 °C, ANGPTL4(E40K) manifested decreased LPL-inhibitory activity compared to ANGPTL4/8, while ANGPTL4(E40K)/8 had even further reduced potency. Remarkably, ANGPTL4/8, ANGPTL4(E40K), and ANGPTL4(E40K)/8 were each actually capable of stimulating LPL activity at 22 °C. Together, these results indicate that ANGPTL4/8 stimulation of LPL activity at low temperatures may represent an additional mechanism for further increasing adipose tissue FA uptake during cold exposure, beyond that already occurring due to decreased ANGPTL4 expression and increased ANGPTL8 expression. In addition, because ANGPTL4(E40K) has decreased LPL-inhibitory activity compared to ANGPTL4/8, our findings also suggest why ANGPTL4(E40K) carriers have decreased circulating TG levels.

Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids.

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Folium nelumbinis (Lotus leaf) volatile-rich fraction and its mechanisms of action against melanogenesis in B16 cells.

This study was aimed at determining the influence of Folium nelumbinis (Lotus leaf) extracts on melanogenesis in vitro models of melanoma cell line. The anticancer activity of four fractions, including petroleum ether (PEE), n-hexane (HE), ethanol (EE), and ethyl acetate (EAE) from F. nelumbinis on B16 cell lines (C57BL/6J melanoma cell), were evaluated after 24 and 48 h treatment. Results showed that PEE as well as volatile-rich fractions of linolenic acid and linolenic acid ethyl ester significantly (p < 0.05) reduced tyrosinase activity and melanin content in B16 melanoma cells model. Meanwhile, PEE and its primarily contained compound triggered apoptosis of B16 cells in a dose-dependent way. These results demonstrated that PEE possessed effective activities against melanin and tyrosinase generations through the induction of apoptosis. Moreover, a relation between the volatile-rich fractions of F. nelumbinis and the anticancer effects was demonstrated as well.

Sonchus oleraceus Linn extract enhanced glucose homeostasis through the AMPK/Akt/ GSK-3ß signaling pathway in diabetic liver and HepG2 cell culture.

The extracts of S. oleraceus Linn (SOL) and its main phenolic compounds have shown anti-diabetic effects, but their underlying mechanisms for glucose homeostasis remain unclear. The aim of this study is to evaluate the anti-diabetic mechanism of SOL by using the streptozocin (STZ) induced diabetic rat model. When diabetic rats were fed with SOL at a dose of 400 mg/kg/day for 6 weeks, the concentrations of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were reduced by 43%, 22%, and 16%, respectively. Meanwhile, it was also found that daily feeding of SOL to diabetic rats led to a decrease in plasma glucose level by approximately 23%. Positive effects were observed on glucose homeostasis due to the down-regulation of AMPK/Akt/GSK-3ß pathway, as indicated by the suppressions of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), protein kinase (Akt) phosphorylation, glycogen synthase kinase 3 beta (GSK-3ß), and the hepatic insulin resistance. In HepG2 cells, AMPK, Akt and GSK-3ß showed a consistent transcript regulation. SOL at dose of 400 mg/kg/day feeding for 6 weeks showed a positive effect comparable to metformin.

Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Citrullination/Methylation Crosstalk on Histone H3 Regulates ER-Target Gene Transcription.

Posttranslational modifications of histone tails are a key contributor to epigenetic regulation. Histone H3 Arg26 and Lys27 are both modified by multiple enzymes, and their modifications have profound effects on gene expression. Citrullination of H3R26 by PAD2 and methylation of H3K27 by PRC2 have opposing downstream impacts on gene regulation; H3R26 citrullination activates gene expression, and H3K27 methylation represses gene expression. Both of these modifications are drivers of a variety of cancers, and their writer enzymes, PAD2 and EZH2, are the targets of drug therapies. After biochemical and cell-based analysis of these modifications, a negative crosstalk interaction is observed. Methylation of H3K27 slows citrullination of H3R26 30-fold, whereas citrullination of H3R26 slows methylation 30,000-fold. Examination of the mechanism of this crosstalk interaction uncovered a change in structure of the histone tail upon citrullination which prevents methylation by the PRC2 complex. This mechanism of crosstalk is reiterated in cell lines using knockdowns and inhibitors of both enzymes. Based our data, we propose a model in which, after H3 Cit26 formation, H3K27 demethylases are recruited to the chromatin to activate transcription. In total, our studies support the existence of crosstalk between citrullination of H3R26 and methylation of H3K27.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain.

BACKGROUND: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. RESULTS: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. CONCLUSIONS: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

A novel CDK9 inhibitor shows potent antitumor efficacy in preclinical hematologic tumor models.

DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.

The 2.5 Å crystal structure of the SIRT1 catalytic domain bound to nicotinamide adenine dinucleotide (NAD+) and an indole (EX527 analogue) reveals a novel mechanism of histone deacetylase inhibition.

The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.

Pharmacogenomics of gemcitabine metabolism: functional analysis of genetic variants in cytidine deaminase and deoxycytidine kinase.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

Crystal structure of the human PRMT5:MEP50 complex.

Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

Development of a novel sandwich ELISA for measuring cell lysate ABCA1 protein levels.

ATP-binding cassette transporter A-1 (ABCA1) mediates the transfer of cellular cholesterol to lipid-poor apolipoproteins. Liver X receptors (LXRs) are regulators of cholesterol homeostasis that increase transcription of ABCA1. Synthetic LXR agonists developed to date have been shown to induce ABCA1 mRNA expression and increase reverse cholesterol transport. Unfortunately, there have been few options for quantitatively measuring ABCA1 protein levels, including a previously described competitive ELISA standardized to an ABCA1 peptide with a sensitivity of 80 ng/ml. To address this unmet need, we developed a novel sandwich ELISA standardized to full-length human recombinant ABCA1 protein with sensitivity of approximately 0.5 ng/ml. To determine if the sandwich ELISA had adequate sensitivity to detect LXR-induced increases in ABCA1, we utilized it to measure ABCA1 levels in untreated and LXR agonist-treated human (THP-1) macrophage cells and human peripheral blood mononuclear cells (PBMC). Data obtained from the ELISA demonstrated an approximately eightfold increase in ABCA1 levels in both macrophages as well as PBMC in response to LXR agonist treatment, and results were highly correlated with those obtained by immunoprecipitation and western blotting. Together, these results suggest that the sandwich ELISA may be a sensitive and effective method for quantitating ABCA1 protein levels.

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 microg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30min and the degradation of hepatic LDLR reached the maximum 2h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5min of protein injection and internalized PCSK9 was only barely detectable within 1h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.

A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Derivation and characterization of monoclonal antibodies against human folypolyglutamate synthetase.

Folypolyglutamate synthetase (FPGS) plays a critical role in the cellular retention of both folates and antifolates. Resistance to antifolates is in part related to changes in FPGS enzyme activity and levels of messenger RNA, or in some instances, protein as evaluated by Western blots using polyclonal antisera. The present study was designed to derive a series of monoclonal antibodies (MAb) against the native protein, to characterize them in terms of specificity and epitope mapping, and to determine kinetic constants by Biacore. We report on 3 IgG(1) kappa MAbs-namely, 4-2, 4-3, and 4-18-with epitopes localized to the carboxyl domain of the protein. These antibodies recognize a single band on Western blots of HeLa cell lysates, which is significantly reduced following RNAi knockdown. The recognition of both the native and denatured conformations of FPGS by these MAbs should provide useful reagents for FPGS quantitation in either tumor cell lysates or in tumor biopsies.

Derivation and characterization of a monoclonal antibody against human glycinamide ribonucleotide formyltransferase.

Glycinamide ribonucleotide formyltransferase (GARFT) is a trifunctional enzyme involved in purine biosynthesis. Its central role in folate metabolism has made it an obvious target for the development of GARFT inhibitors, primarily for oncology. While the crystal structure, enzyme kinetics, and mechanism of action of GARFT inhibitors are reasonably well understood, GARFT regulation at the protein level remains unclear. The present study reports the development and characterization of a monoclonal antibody (MAb) specific for human GARFT. This MAb, an IgG1kappa, designated PHR1, recognizes human GARFT by both Western blot and by immunohistochemistry from non-small-cell lung carcinoma and colon adenocarcinoma tissue biopsies, has a KD of 1.14 x 10(10) M, and has been epitope mapped at residues 59-78 of the GARFT functional domain. The ability of PHR1 to recognize both sodium dodecyl sulfate (SDS)-denatured as well as native GARFT should make this MAb an important research tool in determining GARFT protein levels in both normal and neoplastic tissues.