Joan R. Kanter

Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia

Cyclic nucleotide phosphodiesterase (PDE) isoforms can influence disease pathogenesis and be novel therapeutic targets. Because lower cAMP levels may contribute to the decreased apoptosis that occurs in chronic lymphocytic leukemia (CLL), we assessed the expression levels of PDE isoforms in peripheral blood mononuclear cells (PBMC) of healthy adults and patients with CLL. We found a unique PDE mRNA signature in CLL: higher levels than in normal PBMC of PDE7B (increased ≈23-fold) and lower levels of PDE3B, 4D, 5A, and 9A mRNA (each decreased ≈30-fold). Increased PDE7B mRNA in CLL correlates with a 10-fold-higher expression of PDE7B protein and results in an increased contribution of PDE7 to total PDE activity. Consistent with the higher level of PDE7B expression, inhibitors of PDE7 (BRL-50481, IR-202) and a dual PDE4/PDE7 inhibitor (IR-284) selectively increase apoptosis in CLL cells compared with normal PBMC or B cells. Apoptosis of CLL cells promoted by inhibitors of PDE7 and PDE4/7 is attenuated by PKA inhibition, occurs via a mitochondrial-dependent process, and is associated with increased cAMP accumulation and down-regulation of the antiapoptotic protein survivin and of PDE7B. The increase in PDE7B expression and PDE7 inhibitor-promoted apoptosis implicates PDE7B as a drug target in CLL. Our findings identify a unique PDE signature in CLL and illustrate the utility of broad analyses of PDE isoform expression in human disease.

Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

α-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the α-neurotoxin from Naja mossambica mossambica(Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR α-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of α-neurotoxin fromN. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2–3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6–2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the αδ binding site with observed coupling energies of 1.5–1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the α-subunit region between residues 188–200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/π interactions with Tyr190.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

BACKGROUND The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. RESULTS As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. CONCLUSIONS A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.

Expression and Activity of Mutants of Fasciculin, a Peptidic Acetylcholinesterase Inhibitor from Mamba Venom

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, ΔPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short α-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other α-neurotoxins, binds with high affinity to αγ and αδ subunit interfaces (K D ∼100 pm) but binds with markedly reduced affinity to the αε interface (K D ∼100 nm). By constructing chimeras composed of portions of the γ and ε subunits and coexpressing them with wild type α, β, and δ subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, γPro-175 and γGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in ε, confer low affinity. To identify an interaction between γGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between γGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the γ subunit surface of the binding site interface.

Crystal Structure of Mouse Acetylcholinesterase

In mammals, the molecular forms of the cholinesterases are primary determinants of their tissue distribution and disposition within a cell; association of subunits also governs the turnover of the enzyme. The predominant form of acetylcholinesterase (AChE) in the central nervous system is an amphiphilic tetramer anchored to the membrane via a hydro-phobic, non-catalytic subunit, whereas at the neuromuscular junction it is an asymmetric form containing one to three tetramers, associated with the basal lamina via a collagen-like subunit [cf. 1]. Abnormal associations of AChE arise in dementias of the Alzheimer type [2], where a significant and selective loss of the amphiphilic AChE tetramers is observed [3, 4]. However, there is currently little structural information about the molecular determinants involved in association of subunits into tetramers and of tetramers with membrane-anchor subunits.

Cyclic nucleotide phosphodiesterase 7B mRNA: An unfavorable characteristic in chronic lymphocytic leukemia†

A cost‐ and time‐efficient means to define the prognosis of patients with chronic lymphocytic leukemia (CLL) is desirable but does not yet exist. On the basis of the evidence that CLL cells have enhanced expression of the cyclic nucleotide phosphodiesterase isoform 7B (PDE7B), we hypothesized that PDE7B expression might provide such information. We assessed PDE7B mRNA expression using quantitative real‐time PCR in peripheral blood mononuclear cells isolated from 85 patients and 30 normal subjects. We compared PDE7B mRNA expression with that of other disease features to determine if its expression correlates with the prognosis of patients with CLL. We found that CLL patients with PDE7B mRNA levels in the top quartile (greater than ninefold elevation relative to normal controls) have a several‐year shorter median time‐to‐treatment (TTT, 36 months) compared to that of patients whose CLL cells express lower levels of PDE7B mRNA (TTT, 77 months, p = 0.001). High PDE7B mRNA expression correlates with expression of zeta‐chain‐associated protein kinase 70 (ZAP‐70), unmutated immunoglobulin heavy chain variable (IGHV) region genes and β2 microglobulin (β2M), but use of a multivariate Cox model revealed that high PDE7B mRNA expression independently predicts a short TTT, even after adjusting for several other disease characteristics (ZAP‐70 or CD38 expression, IGHV mutation status and Rai status). High expression of PDE7B is an unfavorable characteristic in CLL. Assessment of PDE7B mRNA expression thus appears to be a clinically useful biomarker to define the prognosis of patients with CLL.

Inhibition of cyclic AMP efflux by insect pheromones and fatty acids

Avian erythrocytes export cyclic AMP by a means that prostaglandins A1 and A2, but not other eicosanoids, inhibit (EC50 ≈ 45 nM). Several insect pheromones and the fatty acyl components of common membrane phospholipids also inhibit cyclic AMP efflux (EC50 ≈ 3O μM). The presence of at least one double bond in the acyl chain enhances the effect. Unlike PGA, fatty acids probably do not act via formation of a glutathione adduct but very likely by altering membrane fluidity. Inhibition of cyclic AMP export provides a mechanism by which products of phospholipid metabolism can influence the cyclic AMP signaling pathway.

Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system

Significance Cyclic AMP, the first identified second messenger, regulates a wide array of cellular functions including apoptosis by activating protein kinase A (PKA) and, in turn, the phosphorylation of target proteins. The current study uses a variety of biochemical and functional analyses to assess wild-type S49 lymphoma cells and kin–, a clonal variant that lacks PKA. The results identify key alterations in the ability of kin– cells to process PKA and also define previously unidentified alterations in cAMP- and glucocorticoid-promoted killing of kin– S49 cells. The findings provide evidence for PKA-dependent pathway switching in cell death responses and have implications for therapeutic development in diseases with aberrant apoptosis. Cyclic AMP/protein kinase A (cAMP/PKA) and glucocorticoids promote the death of many cell types, including cells of hematopoietic origin. In wild-type (WT) S49 T-lymphoma cells, signaling by cAMP and glucocorticoids converges on the induction of the proapoptotic B-cell lymphoma-family protein Bim to produce mitochondria-dependent apoptosis. Kin–, a clonal variant of WT S49 cells, lacks PKA catalytic (PKA-Cα) activity and is resistant to cAMP-mediated apoptosis. Using sorbitol density gradient fractionation, we show here that in kin– S49 cells PKA-Cα is not only depleted but the residual PKA-Cα mislocalizes to heavier cell fractions and is not phosphorylated at two conserved residues (Ser338 or Thr197). In WT S49 cells, PKA-regulatory subunit I (RI) and Bim coimmunoprecipitate upon treatment with cAMP analogs and forskolin (which increases endogenous cAMP concentrations). By contrast, in kin– cells, expression of PKA-RIα and Bim is prominently decreased, and increases in cAMP do not increase Bim expression. Even so, kin– cells undergo apoptosis in response to treatment with the glucocorticoid dexamethasone (Dex). In WT cells, glucorticoid-mediated apoptosis involves an increase in Bim, but in kin– cells, Dex-promoted cell death appears to occur by a caspase 3-independent apoptosis-inducing factor pathway. Thus, although cAMP/PKA-Cα and PKA-R1α/Bim mediate apoptotic cell death in WT S49 cells, kin– cells resist this response because of lower levels of PKA-Cα and PKA-RIα subunits as well as Bim. The findings for Dex-promoted apoptosis imply that these lymphoma cells have adapted to selective pressure that promotes cell death by altering canonical signaling pathways.

Fasciculin Inhibition of Mouse Acetylcholinesterase

Fasciculins are 7 kDa proteins that contain four disulfide bridges and are isolated from mamba venoms. They are members of the superfamily of three-fingered Elapidae snake toxins that include the selective nicotinic acetylcholine receptor blockers, α- and κ-neurotoxins, the subtype-specific muscarinic acetylcholine receptor agonists, termed “muscarinic toxins”, the L-type calcium channel blockers, calciceptine and FS2, the GPIIa-IIIa antagonist and platelet aggregtion inhibitor, RGD-containing dendroaspin (or mambin), and the cell membrane lytic cardiotoxins (or cytotoxins) [1, and references therein]. Despite a highly conserved structural motif, the toxins in this family are directed to diverse targets, yet their individual modes of action are highly selective: fasciculins inhibit mammalian, electric fish, and some snake venom acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range, but micromolar concentrations are required to inhibit avian and insect AChEs, and butyrylcholinesterase [cf. 2 for review].