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The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.

Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.

We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.

Allosteric activation of 5(')-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS(133)-CREB and an engineered peptide ACC-CREBp. Anti-pS(133)-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl-CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS(133)-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody-antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use.

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described., (Copyright 1999 Academic Press.)

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors., (Copyright 1999 Academic Press.)