BACKGROUND OF THE INVENTION
 The invention described herein was made with Government support under Merit Review and Career Development Enhancement Awards from the Department of Veterans Affairs. The Federal Government has certain rights in the invention. This application claims the benefit under 35 U.S.C. §119(e) of provisional U.S. patent application No. 60/309,271, filed Aug. 1, 2001.
1. Field of the Invention
The present invention relates to the field of cardiovascular and other diseases. More particularly, the present invention concerns compositions and methods of identification and use of isoform selective activators or inhibitors of type 3 phosphodiesterase (PDE3). Other embodiments of the invention concern high-throughput screening for novel pharmaceuticals directed against PDE3 isoforms. In certain embodiments, the compositions and methods disclosed herein are of use for treatment of cardiomyopathy, pulmonary hypertension and related conditions.
2. Description of Related Art
PDE3 cyclic nucleotide phosphodiesterases hydrolyze cAMP and cGMP and thereby modulate cAMP- and cGMP-mediated signal transduction (Shakur et al., 2000a). These enzymes have a major role in the regulation of contraction and relaxation in cardiac and vascular myocytes. PDE3 inhibitors, which raise intracellular cAMP and cGMP content, have inotropic effects attributable to the activation of cAMP-dependent protein kinase (PK-A) in cardiac myocytes and vasodilatory effects attributable to the activation of cGMP-dependent protein kinase (PK-G) in vascular myocytes (Shakur et al., 2000a). When used in the treatment of dilated cardiomyopathy, PDE3 inhibitors such as milrinone, enoximone and amrinone initially elicit favorable haemodynamic responses, but long-term administration increases mortality by up to 40% (Nony et al., 1994). This linkage of short-term benefits of PDE3 inhibition to deleterious effects on long-term survival in dilated cardiomyopathy is one of the most perplexing problems in cardiovascular therapeutics. However, it is thought that these biphasic effects reflect the compartmentally-nonselective increases in intracellular cAMP content in cardiac myocytes current inhibitors display.
Clinical trials of the use of β-adrenergic receptor agonists—which, like PDE3 inhibitors, increase intracellular cAMP content in cardiac myocytes—were terminated prior to completion because of increased mortality in treated patients, while β-adrenergic receptor antagonists, which reduce intracellular cAMP content, have been shown to improve long-term survival despite initially adverse haemodynamic effects. These findings suggest that both the short-term benefits and long-term adverse effects of PDE3 inhibition are attributable to increases in intracellular cAMP content in cardiac myocytes (Movsesian, 1999).
The contradictory effects of nonspecific PDE3 antagonists may relate to the diverse intracellular processes regulated by cAMP in cardiac and vascular cells. Upon activation by cAMP, PK-A phosphorylates dozens of proteins in separate intracellular compartments that are involved in contraction and relaxation, glycogen metabolism, gene transcription, intracellular Ca2+ cycling and signal autoregulation. Phosphorylation of cAMP-response element-binding protein (CREB), for example, activates the transcription of genes containing cAMP response elements (Shaywitz and Greenberg, 1999). Transgenic mice expressing a dominant non-phosphorylatable CREB in cardiac myocytes develop a dilated cardiomyopathy that very closely resembles the human disease (Fentzke et al., 1998), suggesting that CREB phosphorylation may be desirable in dilated cardiomyopathy.
Another example of cAMP effects is the phosphorylation of phospholamban, which relieves its inhibition of SERCA2, the Ca2+-transporting ATPase of the sarcoplasmic reticulum (Simmerman and Jones, 1998). Ablation of phospholamban in muscle LIM protein (MLP)−/−mice with dilated cardiomyopathy results in the restoration of normal chamber size and contractility (Minamisawa et al., 1999), suggesting that phospholamban phosphorylation may also be beneficial in cardiomyopathy.
Other substrates phosphorylated by PK-A may contribute to adverse effects on long-term survival. Phosphorylation of L-type Ca2+ channels increases their open probability and may be arrhythmogenic (Fischmeister and Hartzell, 1990), while phosphorylation of proteins in the mitogen-activated protein kinase (MAP kinase) cascade may alter myocardial gene transcription so as to speed the progression of the disease (Cook and McCormick, 1993; Lazou et al., 1994).
Raising cAMP content in cardiac myocytes via mechanisms such as activation of β1-adrenergic, β2-adrenergic or prostaglandin receptors or non-selective phosphodiesterase inhibition by isobutylmethylxanthine, affects cAMP content differentially in intracellular compartments represented in cytosolic and microsomal fractions of cardiac muscle, resulting in different patterns of protein phosphorylation and different physiologic responses (Hayes et al., 1980; Xiao and Lakatta, 1993; Xiao et al., 1994; Rapundalo et al., 1989; Jurevicius and Fischmeister, 1996). These considerations are particularly relevant to the pathophysiology of dilated cardiomyopathy, in which receptor-mediated and receptor-independent reductions in cAMP generation are prominent features (Movsesian, 1999; Lutz, et al., 2001). Comparison of cytosolic cAMP content in cytosolic and microsomal fractions between failing and non-failing hearts shows greater reduction in cAMP content in microsomal fractions of failing myocardium than in cytosolic fractions (Böhm, 1994).
The phosphorylation of individual substrates of PK-A may be differentially regulated in response to extracellular signals. Evidence for differential regulation comes from experiments examining the effects of stimulating adenylate cyclase activity and cAMP formation via β1-adrenergic, β2-adrenergic or PGE1 receptors. Activation of β-adrenergic receptors increases cAMP content in both cytosolic and microsomal fractions of cardiac myocytes and elicits contractile responses, while activation of PGE1 receptors increases cytosolic but not microsomal cAMP content and evokes no contractile response (Hayes et al., 1980; Buxton and Brunton, 1983). Increases in the amplitude of intracellular Ca2+ transients in response to β1-adrenergic receptor activation correlate with changes in microsomal cAMP content and are accompanied by increases in phospholamban phosphorylation. Conversely, activation of β2-adrenergic receptors results in an increase in the amplitude of intracellular Ca2+ transients that does not correlate with changes in microsomal cAMP content and occurs without increases in phospholamban phosphorylation (Hohl and Li, 1991; Xiao et al., 1993, 1994). Thus, activation of different receptors linked to cAMP metabolism can elicit different responses in cardiac tissues.
β-adrenergic receptor stimulation and nonselective phosphodiesterase inhibition have different effects on cAMP-activated protein phosphorylation in cardiac myocytes (Rapundalo et al., 1989; Jurvicius and Fischmeister, 1996) that are relevant to the pathophysiology of dilated cardiomyopathy. In that condition, a downregulation of β1-adrenergic receptors and an uncoupling of β-adrenergic receptor occupancy and adenylate cyclase stimulation (attributable to increases in β-adrenergic receptor kinase, Gαi and nucleoside diphosphate kinase) contribute to an impairment in cAMP generation (Movsesian, 1999; Lutz et al., 2001). Studies of cAMP content in cytosolic and microsomal fractions of failing and nonfailing hearts demonstrate a far greater reduction in cAMP content in microsomal fractions than in cytosolic fractions of failing myocardium (Böhm et al., 1994). Taken together, these results indicate that cAMP content in different intracellular compartments can be selectively regulated to invoke different responses reflecting the phosphorylation of different substrates of PK-A. Further, this regulation is altered in dilated cardiomyopathy.
Different isoforms of PDE3 are expressed in cardiac and vascular myocytes and are localized to different intracellular compartments. The different PDE3 isoforms may differ in their regulation by PK-A and PK-B (protein kinase B, also known as Akt). PK-B, a downstream effector of insulin-like growth factors, is an anti-apoptotic mediator in cardiac myocytes (Fujio et al., 2000; Matsui et al., 1999; Wu et al., 2000). PK-B may also be involved in proliferative responses in vascular myocytes (Rocic and Lucchesi, 2001; Duan et al., 2000; Sandirasegarane et al., 2000). These findings suggest that different PDE3 isoforms may be involved in cell- and compartment-selective responses to different signals that have been implicated in the pathophysiology of dilated cardiomyopathy and/or pulmonary hypertension. Different PDE3 isoforms in cardiac and vascular myocytes may regulate functionally distinct pools of cAMP and cGMP involved in the phosphorylation of different substrates of PK-A and PK-G, and these isoforms may be regulated in response to different extracellular signals.
- SUMMARY OF THE INVENTION
Until the present invention, it was not possible to develop isoform selective inhibitors or activators of PDE3 to use in the treatment of cardiomyopathy and/or pulmonary hypertension. Isoform selective PDE3 inhibitors may provide a beneficial effect on cardiac output without the long term mortality associated with non-specific PDE3 inhibitors. Isoform-selective PDE3 activators may have beneficial anti-apoptotic effects in patients with dilated cardiomyopathy and/or pulmonary hypertension whose hemodynamic status is not too compromised to tolerate a reduction in cardiac contractility, without concomitant arrhythmogenic effects attributable to increases in cytosolic cAMP content. A paradigm for the latter is the use of beta-adrenergic receptor antagonists in the treatment of dilated cardiomyopathy.
Agents capable of selectively activating or inhibiting individual PDE3 isoforms or of disrupting their intracellular localization may selectively affect the phosphorylation of smaller subsets of PK-A and PK-G substrates to therapeutic advantage. Without wishing to be limited to any one specific embodiment, an agent that selectively inhibits sarcoplasmic reticulum-associated PDE3A-136 may help to preserve intracellular Ca2+ cycling and contractility in patients with dilated cardiomyopathy taking β-adrenergic receptor agonists, which may reduce arrhythmogenic effects attributable to increases in cytosolic cAMP content. Alternatively, if the activation of PDE3A-136 by PK-B is anti-apoptotic in cardiac myocytes, its inhibition may be pro-apoptotic (possibly explaining the increased long-term mortality seen with PDE3 inhibition in dilated cardiomyopathy), and the selective activation of this isoform may be desirable. In addition, currently available competitive PDE3 inhibitors inhibit cAMP activity more potently than they inhibit cGMP hydrolytic activity, owing to the higher Km's of the hydrolytic enzymes for cAMP than for cGMP. Agents that inhibit PDE3 activity through other mechanisms, identified by the methods described herein, may affect hydrolysis of the two substrates differentially, resulting in different cellular actions of therapeutic benefit.
As disclosed herein, N-terminal differences exist between the different isoforms of PDE3. Without wishing to be limited to any one specific embodiment, these N-terminal differences may offer opportunities for targeting individual isoforms of PDE3. Differences with respect to phosphorylation sites that stimulate catalytic activity suggest that agents that bind to domains containing these sites so as to either block phosphorylation or mimic its effects may be useful as isoform-selective PDE3 inhibitors or activators. As an example, an agent that binds to the P1 phosphorylation site could selectively inhibit or activate PDE3A-136 or PDE3B-137. A similar rationale would apply to agents that bind to N-terminal protein-interacting domains so as to either block or mimic the effects of these interactions, with the paradigm of peptides that modulate cAMP-mediated signaling by blocking PK-A/AKAP interactions (Rosenmund, et al., 1994). Without wishing to be limited to any one specific embodiment, the typical accessibility of phosphorylation sites and protein interacting domains makes them propitious drug targets. Differences between PDE3A and PDE3B in the N-terminal regions are sufficient to permit selective targeting of PDE3A-136 v. PDE3B-137, which may allow selective modulation of PDE3 activity in cardiac and vascular myocytes.
As shown herein, the different isoforms of PDE3 are translated from different mRNAs. In some cases these mRNAs are generated from different genes (PDE3A and PDE3B). In the case of PDE3A, different isoforms are generated from different mRNAs transcribed from the same gene (e.g., PDE3A1 and PDE3A2 mRNAs). The open reading frame (ORF) of PDE3A1 is indicated in SEQ ID NO:14. The 5′ untranslated region (5′-UTR) of PDE3A1, starting with the first ATG codon, is listed in SEQ ID NO:18. The approximate ORF of PDE3A2 is indicated in SEQ ID NO:15. A nucleotide sequence unique to PDE3A1 mRNA has been identified, and cDNA probes have been designed that react with PDE3A1 mRNA but not PDE3A2 mRNA. Without wishing to be limited to any one specific embodiment, these differences make PDE3 mRNAs propititious targets for decreasing the activity of individual protein isoforms by inhibiting the translation of their mRNAs via antisense constructs, ribozymes or small interfering RNA's (“siRNAs”).
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention fulfills an unresolved need in the art by identifying differences between PDE3 isoforms that may be used to develop isoform selective inhibitors or activators of PDE3 activity. Such inhibitors or activators are proposed to allow the differential regulation of cAMP and cGMP levels in different subcellular compartments, cell types and tissues. In certain embodiments, the present invention concerns methods for identifying isoform selective PDE3 inhibitors or activators. Certain embodiments concern compounds identified by such methods that are of use for the therapeutic treatment of cardiomyopathy and/or pulmonary hypertension. In preferred embodiments, such compounds result in improved cardiac output while exhibiting little or no long-term toxicity. In other embodiments, the isoform selective inhibitors or activators of PDE3 find utility for therapeutic treatment of a number of disease states related to defects in the regulation of cAMP concentration, such as diabetes mellitus, peripheral vascular disease and coronary artery stenosis (especially—but not limited to—stenoses occurring after coronary angioplasty).
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 Role of PDE3 in cAMP- and cGMP-mediated signal transduction. PK-A: cAMP-dependent protein kinase; PK-G: cGMP-dependent protein kinase.
FIG. 2 PK-A substrates in cardiac myocytes. AKAP: PK-A (‘A kinase’) anchoring protein; CREB: cAMP response element-binding protein; Gly Syn: glycogen synthase; Ph K: phosphorylase kinase; PI3-K: phosphatidylinositol 3-kinase; PL: phospholamban; Ry: ryanodine; SERCA: Sarcoplasmic/endoplasmic reticulum calcium ATPase; Tn: troponin; TM: tropomyosin.
FIG. 3 Generation of PDE3A mRNA's by alternative transcription. Shaded boxes represent exons of PDE3A.
FIG. 4 Functional topography of PDE3A and PDE3B open reading frames showing NHR1 and 2, CCR with INS and PK-B and PK-A sites (‘B’, ‘Aup’ and ‘Ad:’). Numbers between dotted lines denote % amino acid sequence identities of homologous regions.
FIG. 5A Western blotting of rtPDE3A1 (containing the full ORF product of PDE3A1) and microsomal and cytosolic fractions of human myocardium. A lysate of Sf21 cells expressing a full-length open reading frame ORF of rePDE3A1 (1.0 μg/lane) and microsomal and cytosolic fractions of human myocardium (50 and 20 μg/lane, respectively) were subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose membranes and Western blotting, using anti-NT, anti-MID and anti-CT antibodies.
FIG. 5B Location of anti-NT, anti-MD and Anti-CT binding sites on the full-length ORF of PDE3A1.
FIG. 6 Comparison of molecular weights of [35S]-labeled rtPDE3A proteins, showing SDS-PAGE autoradiograms, and native cardiac and aortic isoforms of PDE3A, identified by Western blotting of membranes prepared from the same gels with antibodies as indicated. The numbers below the autoradiograms indicate the initial start codon of the PDE3A derived construct.
FIG. 7 Generation of cardiac and aortic isoforms of PDE3A. PDE3A1 and PDE3A2 mRNA's were generated by alternative transcription. PDE3A1 is expressed only in cardiac myocytes. PDE3A2 is expressed in both cardiac and aortic myocytes. PDE3A-136 is translated from PDE3A1. PDE3A-118 and PDE3A-94 are translated from alternative sites in PDE3A2. Numbers in “mRNA” refer to start codons. P1, P2 and P3 designate phosphorylation sites.
FIG. 8 Inhibition of cAMP hydrolytic activity of rtPDE3A1 (in Sf9 lysates) and cytosolic and microsomal fractions of human myocardium by milrinone.
FIG. 9 Stimulation of cGMP hydrolytic activity by PK-A. Detergent solubilized lysates of Sf21 cells expressing rtPDE3B isoforms (full-length ORF's, including wild-type, Ser→Ala and Ser→Asp; mutations) were prepared, and cGMP hydrolytic activity was determined at 0.03 μM cGMP after incubation in the presence or absence of PK-A and ATP. Values represent mean ± standard deviation (each pair of values represents data from a single preparation).
FIG. 10 Co-immunoprecipitation of rtPK-B and rtPDE-B. Amino acid sequences of rtPDE3B are shown at top. Detergent-solubilized lysates of Sf9 cells infected with rtPDE3B were mixed with lysates from Sf9 cells infected with rtPK-B. Proteins were immunoprecipitated with anti-PDE3B antibodies and subjected to Western blotting with anti-PDE3B and anti-PK-B antibodies. PK-B coprecipitates with the full-length but not the truncated rtPDE3B. The identity of the 92 kDa band is unknown.
FIG. 11 Open reading frame of PDE3A (see SEQ ID NO:14). The apparent N-terminal methionine residues of the three isoforms are indicated in bold for PDE3A-136 (amino acid 146), PDE3A-118 (amino acid 300) and PDE3A-94 (amino acids 484 or 485). The phosphorylation sites on the PDE3A isoforms are indicated by underlining for P1 (amino acids 288-294), P2 (amino acids 309-312) and P3 (amino acids 435-438).
- DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
FIG. 12. Comparison of apparent molecular weights of [35S]rtPDE3A proteins and native cardiac and aortic isoforms of PDE3A. rtPDE3A isoforms were generated by in vitro transcription/translation from constructs with 5′ deletions designed to result in tranlation from different in-fram AUG codons in the PDE3A1 ORF.
The following abbreviations are used herein. Other abbreviations not listed below have their plain and ordinary meaning.
AKAP: PK-A (‘A kinase’) anchoring protein
Akt: protein kinase B
anti-CT: a polyclonal antibody raised against the C-terminus of PDE3A
anti-MID: a polyclonal antibody raised against a mid-protein amino acid sequence in PDE3A
anti-NT: a polyclonal antibody raised against the N-terminus of PDE3A
CCR: conserved catalytic region
CK2: casein kinase 2
CREB: cAMP response element-binding protein
G: G protein (Gα, Gβ, Gγ)
Gly Syn: glycogen synthase
IGF: insulin-like growth factor
INS: 44-amino acid insert in CCR
MAP kinase: mitogen-activated protein kinase
MLP: muscle LIM protein
NHR: N-terminal hydrophobic region
p34cdc2: cyclin-dependent proteinkinase
P1, P2, P3: phosphorylation sites in PDE3
PDE3: type 3 phosphodiesterase
PDE3-BP: PDE3-binding protein
PGE1: prostaglandin E1
Ph K: phosphorylase kinase
PI3-K: phosphatidylinositol 3-kinase
PK-A: cAMP-dependent protein kinase
PK-B: protein kinase B, also known as Akt
PK-C: protein kinase C
PK-G: cGMP-dependent protein kinase
PKI: a protein kinase inhibitor specific for PK-A
RACK: receptor for activated PK-C
rtX: recombinant form of protein ‘X’
SERCA: Sarcoplasmic/endoplasmic reticulum calcium ATPase
V8: endopeptidase Glu-C
As used herein, “a” or “an” may mean one or more than one of an item.
This application concerns, at least in part, isolated proteins and nucleic acids encoded by type 3 phosphodiesterase (PDE3, GenBank Accession No. NM000921), as well as methods of identification of isoform selective inhibitors or activators and methods of therapeutic treatment of cardiomyopathy and/or pulmonary hypertension directed towards such proteins. In the present disclosure, reference to “PDE3” or “type 3 phosphodiesterase” without further qualification or limitation means any or all of the isoforms of PDE3, either identified herein or as discovered or characterized by the methods disclosed herein. Where the sequences of the disclosed PDE3A isoforms proteins (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) differ from the GenBank sequence, the sequences disclosed herein are believed to be more accurate and are preferred.
A “PDE3 isoform” is a variant of type 3 phosphodiesterase that differs in its primary structure (i.e., amino acid sequence) from other isoforms of PDE3. The term encompasses, but is not limited to, isoforms that are produced by truncation, amino acid substitution (mutation) or by alternative mRNA splicing, so long as some difference in amino acid sequence results. For the purposes of the present invention, other types of covalent modification would be considered to fall within the scope of a single isoform. For example, both phosphorylated and unphosphorylated forms of PDE3A-136 would be considered to represent the same isoform. The amino acid sequences of the three isoforms of PDE3A are as disclosed in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
As used herein, an “inhibitor” of PDE3 means any compound or combination of compounds that acts to decrease the activity of PDE3, either directly or indirectly, with respect to catalyzing the breakdown of cAMP and/or cGMP. An inhibitor can be a molecule, an atom, or a combination of molecules or atoms without limitation. The term “antagonist” of PDE3 is generally synonymous with an “inhibitor” of PDE3. Inhibitors may act directly on PDE3 by, for example, binding to and blocking the catalytic site or some other functional domain of PDE3 that is required for activity. An inhibitor may also act indirectly, for example, by blocking the phosphorylation (or its effect on activity) or facilitating the dephosphorylation of PDE3 or by facilitating or interfering with the binding of PDE3 to another protein or peptide. The skilled artisan will realize that inhibitors and/or activators may affect PDE3 isoform protein activity and/or may affect the transcription, processing, post-transcriptional modification, stability and/or translation of one or more mRNA species encoding PDE3 isoform proteins (see, e.g., GenBank Accession No. NM000921, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18).
As used herein, an “activator” of PDE3 means any compound or combination of compounds that acts to increase the activity of PDE3, either directly or indirectly, with respect to catalyzing the breakdown of cAMP and/or cGMP. An activator can be a molecule, an atom, or a combination of molecules or atoms without limitation. The term “agonist” of PDE3 is generally synonymous with an “activator” of PDE3. Activators may act directly on PDE3 by, for example, binding some functional domain of PDE3 that is required for activity or by altering the secondary, tertiary or quaternary structure of PDE3 in a way that increases activity. An activator may also act indirectly, for example, by facilitating the phosphorylation or mimicking its effect, by blocking the dephosphorylation of PDE3 or by facilitating or interfering with the binding of PDE3 to another protein or peptide. As discussed above, activators may affect PDE3 isoforms at the level of mRNA and/or protein.
- Type 3 Phosphodiesterase
An “isoform selective” inhibitor or activator of PDE3 is one that has a greater effect on one isoform of PDE3 than on any other isoform of PDE3. In preferred embodiments, an “isoform selective” inhibitor or activator has at least a two-fold greater, more preferably three-fold greater, more preferably four-fold greater, more preferably five-fold, more preferably ten-fold or more greater effect on one isoform of PDE3 than on any other isoform of PDE3. For purposes of the present invention, the precise degree of selectivity of an inhibitor or activator for one isoform of PDE3 compared to other isoforms is not significant, so long as a desired therapeutic effect is achieved. For example, a desired therapeutic effect might be an improvement in cardiac output, with a decrease in long-term mortality, resulting from administration of an isoform selective PDE3 inhibitor or activator compared with nonspecific PDE3 inhibitors. An “isoform selective” inhibitor or activator of PDE3 encompasses, but is not limited to, an isoform specific inhibitor or activator of PDE3. An isoform specific inhibitor or activator of PDE3 is one that acts almost exclusively upon a single isoform of PDE3, so that the effect of the inhibitor or activator on one isoform of PDE3 compared to any other PDE3 isoform is at least an order of magnitude greater, more preferably two orders of magnitude greater, more preferably three orders of magnitude or more greater.
Cyclic nucleotide phosphodiesterases have a ubiquitous role in regulating cAMP- and cGMP-mediated intracellular signaling. Eleven families of these enzymes have been identified. Those in the PDE3 family are dual-specificity phosphodiesterases that bind both cAMP and cGMP with high affinity and hydrolyze them in a mutually competitive manner (FIG. 1). PDE3 inhibitors, which raise intracellular cAMP and cGMP content, have inotropic effects attributable to the activation of cAMP-dependent protein kinase (PK-A) in cardiac myocytes and vasodilatory effects attributable to the activation of cGMP-dependent protein kinase (PK-G) in vascular myocytes.
In addition to regulating contraction and relaxation in cardiac and vascular myocytes, PDE3 cyclic nucleotide phosphodiesterases are involved in platelet aggregation, anti-lipolytic responses to insulin in adipocytes, insulin secretion by pancreatic β cells and maturation of oocytes (Shakur et al., 2000a; Zhao et al., 1998; Andersen et al., 1998). FIG. 2 illustrates the numerous targets and the intracellular compartmentation of PK-A activity.
Two subfamilies of PDE3, products of genes designated PDE3A and PDE3B, have been identified. PDE3A is expressed primarily in cardiac and vascular myocytes and platelets, while PDE3B is expressed primarily in adipocytes, hepatocytes and pancreatic cells (but also in vascular myocytes) (Reinhardt et al., 1995). To date, one PDE3B (Taira et al., 1993) and three PDE3A cDNAs have been cloned. The latter are generated by transcription from alternative start sites in PDE3A. PDE3A1 (SEQ ID NO:14, SEQ ID NO:18), which was cloned from human myocardium, incorporates all sixteen exons of PDE3A (Meacci et al., 1992; Kasuya et al., 2000). PDE3A2 (SEQ ID NO:15), which was cloned from aortic myocytes, is transcribed from a start site in exon 1 (Choi et al., 2001). PDE3A3, cloned from placenta, is transcribed from a start site between exons 3 and 4 (Kasuya et al., 1995). The alternative start sites used for transcription of the three PDE3A mRNAs are illustrated in FIG. 3. The encoded amino acid sequences of the PDE3A isoforms are disclosed herein as SEQ ID NO:1 (PDE3A-136), SEQ ID NO:2 (PDE3A-118) and SEQ ID NO:3 (PDE3A-94). The skilled artisan will realize that the protein isoforms of PDE3 do not precisely correspond to the mRNA species transcribed from the PDE3A gene. For example, both PDE3A-118 and PDE3A-94 are translated from the PDE3A2 mRNA (SEQ ID NO: 15).
The functional topographies of the proteins corresponding to the longest open reading frames (ORF's) of PDE3A and PDE3B are similar (FIG. 4). The C-terminus includes a sequence of about 280 amino acids, designated as “CCR” (FIG. 4), which is highly conserved among cyclic nucleotide phosphodiesterase families and in which catalytic activity resides. Within CCR lies a 44-amino acid insert, designated “INS”, that is unique to the PDE3 family of cyclic nucleotide phosphodiesterases. The N-terminus contains two hydrophobic sequences, designated “NHR1” (about 200 amino acids) and “NHR2” (about 50 amino acids). NHR1 and NHR2 appear to be implicated in intracellular targeting. Between NHR1 and NHR2 are sites phosphorylated by PK-A and PK-B that, despite their distance from CCR, modulate catalytic activity. A second PK-A site whose function is unclear is located between NHR2 and CCR.
- Structure/Function Relations
Despite the structural similarities, there are considerable differences between PDE3A and PDE3B with respect to their amino acid sequences. PDE3A and PDE3B are 84-86% identical within the CCR region, exclusive of INS. However, INS and the extreme C-terminus are only 35-39% identical, and the remaining upstream regions are less than 30% identical. Thus, while the catalytic sequences of the isoforms are similar, the regulatory portions of the isoforms appear to be very different and are likely to be differentially affected by the various inhibitors and activators of the present invention.
Catalytic activity. The catalytic activity of PDE3 enzymes requires almost the entire C-terminal sequence downstream of about amino acid 650, including the CCR domain that is largely conserved among all PDE families, as well as the INS and the CCR-flanking regions that are unique to the PDE3 family (FIG. 4). (Cheung et al., 1996; He et al., 1998.) The recent determination of the crystal structure of the related enzyme PDE4B2B has led to the identification of its catalytic site (Xu et al., 2000). The catalytic domain consists of three subdomains comprising 17α-helices.
The active site, preserved in all PDE families, is at the junction of these three subdomains and is formed by the apposition of discontinuous amino acids. Differences in substrate affinity and selectivity among isoform families may be influenced in large part by differences in amino acid sequences that allosterically affect Glu1001 of PDE3A, which “reads” the 1- and 6-positions of the cyclic nucleotide purine ring and determines affinity (and hence selectivity) for cAMP and cGMP. Experiments involving PDE3/PDE4 chimeras indicate that the regions adjacent to this site contain the determinants of sensitivity to phosphodiesterase inhibitors (Atienza et al., 1999). This model, in which the active site is formed by discontinuous domains with allosteric determination of substrate affinity, may explain why so much sequence is required for catalytic activity. It may also explain why mutations of some amino acids preferentially affect binding of either cAMP or cGMP, while others affect the binding of both nucleotides (Zhang and Colman, 2000). While the N-terminus is not required for catalytic activity, N-terminal deletions increase the ratio of Vmax cGMP/Vmax cAMP, suggesting that the N-terminal region is involved in regulating catalytic activity (Tang et al, 1997).
The structural model described above has important implications regarding the feasibility of selective PDE3 inhibition or activation. The sequences of regions required for catalytic activity—INS and the regions flanking CCR—differ sufficiently between PDE3A and PDE3B to be reasonable targets for isoform-selective inhibitors or activators. As described in the Examples below, the development of anti-peptide antibodies selective for the C-terminus of either PDE3A or PDE3B is further evidence that selective inhibition or activation may occur. The existence of allosteric sites that differentially affect cAMP and cGMP hydrolysis allows for the identification of small molecules that selectively bind to these sites and affect either cAMP or cGMP hydrolysis.
Intracellular localization. Intracellular targeting of PDE3 appears to be determined principally by the N-terminal domains NHR1 and NHR2. NHR1 contains six transmembrane helices, the last two of which are sufficient to localize recombinant proteins containing these domains exclusively to intracellular membranes (Kenan et al, 2000; Shakur et al., 2000b). Such recombinants can be solubilized only by a combination of high salt and detergent, suggesting that they are intrinsic membrane proteins. Recombinants lacking NHR1 but retaining NHR2 are found in both microsomal and cytosolic fractions of transfected cells. High salt alone is sufficient to solubilize these proteins, suggesting that interactions with other proteins are involved in their intracellular localization. Recombinants lacking both NHR1 and NHR2 are predominantly cytosolic.
Regulation by protein phosphorylation. Phosphorylation of PDE3 plays a major role in the regulation of its function. In adipocytes, phosphorylation of PDE3 by PK-A and perhaps PI3-K are involved in the anti-lipolytic response to insulin (Smith et al., 1991). In oocytes, phosphorylation by PK-B results in the resumption of meiosis (Zhao et al., 1998). In promyeloid cells, phosphorylation by PK-B regulates cAMP pools that modulate DNA synthesis (Ahmad et al., 2000). In platelets, phosphorylation of PDE3A by PK-A and an insulin-activated protein kinase is associated with inhibition of aggregation (Grant et al, 1988; Lopez-Aparicio et al., 1993).
As described in more detail in the Examples below, three phosphorylation sites have been identified for the PDE3 isoforms (FIG. 4). PDE3B is phosphorylated in vivo by PK-A and possibly by PI3-K at Ser318 (site P2) (Rahn et al., 1996; Rondinone et al., 2000). The P2 site is dephosphorylated by a PP2A serine/threonine phosphatase (Resjö et al., 1999). PDE3B is also phosphorylated in vivo by PK-B at Ser296 (site P1) (Kitamura et al., 1999). Phosphorylation at either site increases catalytic activity. The fact that P1 and P2 lie between NHR1 and NHR2 raises the possibility that phosphorylation at these sites also affects intracellular targeting.
A third site—Ser421 in PDE3B (site P3)—is phosphorylated by PK-A in vitro (Rascón et al., 1994). In adipocytes it is unclear whether PDE3B is phosphorylated at P3 in response to isoproterenol or insulin in vivo. It is unknown whether this site is phosphorylated in PDE3B in other cell types and, if so, how phosphorylation at this site affects activity. It is also unknown whether phosphorylation at any of these sites affects inhibitor sensitivity, but a relevant paradigm is the reduction in the sensitivity of another phosphodiesterase, PDE4D3, to the inhibitor rolipram that results from phosphorylation of PDE4D3 by PK-A (Hoffmann et al., 1998). Prior to the present invention, the phosphorylation sites on the PDE3A isoforms were unknown. Numerous consensus phosphorylation sites are present in the PDE3A amino acid sequence and it was unknown which of these sites was phosphorylated in vivo.
- Protein-Protein Interactions
The identification of protein kinases that phosphorylate PDE3 isoforms and alter their function may elucidate their role in dilated cardiomyopathy. Phosphorylation and activation of PDE3 by PK-B, for example, may be an anti-apoptotic mechanism related to the deleterious long-term effects of PDE3 inhibition in dilated cardiomyopathy. The sequences of PDE3A and PDE3B contain multiple consensus sites for CK2, PK-C and other protein kinases. It may be especially important to consider cross-regulation by these kinases in the pathophysiology of cardiomyopathy and/or pulmonary hypertension. By analogy, PDE4D3 phosphorylation by ERK2 profoundly reduces its activity, and this reduction is reversed by phosphorylation by PK-A (Hoffmann et al. 1999).
Interactions with other proteins are involved in the regulation of activity and intracellular localization of other families of PDE. Binding of Ca2+/CaM stimulates catalytic activity of PDE1 via multiple CaM-binding domains (Sonnenburg et al., 1995). The activities of PDE6αβ and α′α′ dimers are inhibited by their interaction with PDEγ. Phototransduction occurs when this inhibition is relieved by interaction with the rhodopsin-coupled G protein transducin (Granovsky et al., 2000). PDE6 dissociates from intracellular membranes upon binding to PDE8 (Florio et al., 1996). Interactions with RACK1 and AKAP's are involved in the subcellular targeting of PDE4 isoforms to multienzyme complexes (Yarwood et al. 1999; Dodge et al., 2001). The interactions of PDE4 with SH3 domains of SRC family tyrosine kinases affect intracellular localization and inhibitor sensitivity (McPhee et al., 1999).
Prior to the present invention, it was unknown whether PDE3 is catalytically regulated or intracellularly targeted via interactions with other proteins. PDE3B, insulin receptor, the p85 and p110subunits of PI3-K and an unidentified 97-kDa protein are co-immunoprecipitated from human adipocytes with anti-insulin receptor antibodies (Rondinone et al., 2000). Preliminary data on the interaction of PDE3B with 14-3-3 proteins has been reported (Palmer et al., 2000). 14-3-3 proteins bind to phosphorylated serine residues in consensus motifs and affect intracellular localization of proteins in diverse ways (Fu et al., 2000). As discussed in the Examples below, site P1 in PDE3A and PDE3B approximates this consensus motif, raising the possibility that phosphorylation affects intracellular localization through interaction with 14-3-3 proteins. The Examples further show the existence of stable complexes of PDE3B with PK-B and AKAP220. Taken together, these observations indicate that interactions of other proteins with the N-terminus are involved in PDE3 function, and that phosphorylation of PDE3 may affect these interactions.
In referring to the function of PDE3 or “wild-type” activity, it is meant that the molecule in question has the ability to catalyze the breakdown of cAMP and cGMP. Molecules possessing this activity may be identified using assays familiar to those of skill in the art. For example, in vitro assay of homogenates containing PDE3 activity, or variants thereof, will identify those molecules having PDE3 activity by virtue of their ability to degrade cAMP or cGMP. The skilled artisan will realize that a variety of phosphodiesterases are endemic to various cell lines and tissues and will select an appropriate system lacking endogenous phosphodiesterase to perform such assays.
The term “PDE3 gene” refers to any DNA sequence that is substantially identical to a DNA sequence encoding a PDE3 protein as defined above. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90%, preferably at least about 95%, most preferably 98% or more of nucleotides that are identical to the cDNA sequences of PDE3 are “as set forth in” those sequences. Sequences that are substantially identical or “essentially the same” as the cDNA sequences of PDE3 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of the cDNA sequences of PDE3 under conditions of relatively high stringency. Such conditions are typically relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. Such selective conditions tolerate little, if any, mismatch between the complementary stands and the template or target strand. Any such gene sequences may also comprise associated control sequences.
In certain embodiments, the present invention relates to fragments of PDE3 polypeptides that may or may not retain the phosphodiesterase activity of PDE3, although in preferred embodiments the fragments exhibit phosphodiesterase activity. Fragments including the N-terminus of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the protein molecule with proteolytic enzymes can produce a variety of N-terminal, C-terminal and internal fragments. Examples of fragments may include contiguous residues of the PDE3 amino acid sequences of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500 or more amino acids in length. These fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography), or various size separations (e.g., sedimentation, gel electrophoresis, gel filtration).
- Protein Purification
Substantially identical analog proteins will be greater than about 80% identical, more preferably 90% identical, more preferably 95% identical, more preferably 98% identical, more preferably 99% identical, more preferably 99.5% identical, more preferably 99.9% identical to the corresponding sequence of the native protein. Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents. In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequence.
Certain embodiments may involve purification of one or more individual PDE3 isoforms or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even HPLC.
Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The terms “isolated” or “purified” as applied to a protein or peptide, are intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide, therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a “−fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like, or by heat denaturation, followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “−fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will, therefore, be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with high resolution of peaks. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a type of partition chromatography that is based on molecular size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size of the pores. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. In gel chromatography, separation is independent of all other factors such as pH, ionic strength, temperature, etc.
- Synthetic Peptides
Affinity chromatography relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. The column material is synthesized by covalently coupling one of the binding partners, such as an antibody or an antibody-binding protein to an insoluble matrix. The column material is then able to specifically adsorb the target substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.). One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
- Two-dimensional Mapping
In some embodiments, the present invention concerns smaller peptides for various uses, such as antibody generation or screening for potential inhibitors or activators that can bind to various epitopes of PDE3. Smaller peptides of about 100 amino acids or less can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automated peptide synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to selected regions of the PDE3 protein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides or other small molecules. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression. Expression of cloned PDE3 sequences is preferred in embodiments where PDE3 peptides of greater than about 50 amino acids in length are desired. The skilled artisan will realize that it is also possible to synthesize short peptide fragments and covalently link them together, for example using carbodiimides as cross-linking groups. In this manner, a peptide of any desired length can be produced by synthesizing shorter fragments and joining them in the appropriate order.
Two dimensional mapping, also known as proteome analysis, is a useful tool for characterization of cellular protein expression. Specifically contemplated are the methods described in Gibson, 1974; Beemon and Hunter, 1978; and Luo, et al., 1990, each of which is incorporated herein by reference, in their entirety. Two-dimensional mapping is based on two-dimensional electrophoretic separation of proteins in a cellular lysate or homogenate so that each protein can be identified using specific coordinates in a two-dimensional protein map from which it can be extracted and further identified (by, e.g., micro sequencing or mass spectrometry).
For mapping, the proteins in a cellular homogenate or lysate are immunoprecipitated, using an antibody or series of antibodies specific for the proteins of interest, and run on a preparative electrophoretic protein gel. The proteins from this gel are then transferred to an immobilizing matrix. Various immobilizing matrices are available and may be used. Preferred matrices for purposes of the present invention are nitrocellulose or a nylon matrix such as Immobilon (Millipore, Bedford, Mass.). The resulting protein-matrix hybrid, called a blot, is then washed with water in order to remove any non-bound cellular debris from the initial homogenate or lysate, which may cause interference in subsequent steps. The blot is then contacted with an antibody, or series of antibodies, specific to the protein or proteins of interest in the cellular homogenate or lysate. The skilled artisan will realize that these antibodies may be monoclonal, polyclonal, or both and use of any will not substantially change the outcome of this procedure. Once the protein or proteins of interest from the cellular homogenate or lysate ore identified by the antibodies, by the antibodies binding to the proteins and forming an antibody-protein complex, they are physically excised from the rest of the blot matrix. One of reasonable skill in the art will recognize that any common method of antibody detection may be used to identify the aforementioned antibody-protein complex. These may include, but are not limited to, ELISA, alkaline-phosphatase conjugated secondary antibody, enzyme-conjugated antibodies, radiolabeled antibodies, or any other common method of detection. For purposes of the present invention, radiolabeled antibodies are the preferred method of detection.
The protein or proteins, still in the form of bands from the immobilizing matrix, are digested by one of several common peptidase enzymes. These are enzymes that cleave proteins at specific locations only and include, but are not limited to, trypsin, chymotrypsin, CNBr and V8. Digestion may be allowed to run to completion, i.e. where every possible site that the chosen peptidase could recognize in the sample is cleaved, or it may be a partial digestion, merely run for a shorter period of time and not to completion. Once the desired level of digestion is completed, the peptidase chosen is removed from the sample, typically by centrifugation and transfer of the supernatant to a new container or vessel.
These digested samples are then loaded onto a cellulose thin layer plate for pH-driven electrophoresis, the first “dimension” in the mapping process. The digested proteins will behave on this thin layer plate much as they would when subjected to standard SDS-PAGE, except that the digested protein fragments will separate by charge according to the pH of the electrophoresis buffer. By way of example only, if the electrophoresis buffer chosen has a pH ranging from 1.9 to 4.72, then the majority of the digested peptide fragments in the sample will be positively charged. The thin layer plate should thus be loaded appropriately for optimal separation o the digested peptide fragments. In this example, the plate should be loaded at a distance closer to the positive electrode and farther from the negative electrode. The skilled artisan will recognize that the pH used in any individual electrophoresis should be that which will give an optimal distribution o the peptides. Preferred pH values include 8.9, more preferably 4.72, even more preferably 1.9. After electrophoresis is complete, the thin layer plate is typically dried in an oven. It is thought that this step irreversibly binds the digested peptide fragments to the cellulose on the thin layer plate.
Chromatography, the second “dimension” in the mapping, is next performed. The thin layer plate is placed in a chamber with a chromatography liquid, but only one side of the thin layer plate is immersed in this liquid. The thin layer plate should be placed in the liquid in such a manner that the liquid used, as it travels up through the thin layer plate via capillarity, does so at a ninety (90) degree angle from the direction electrophoresis was performed on the plate. When chromatography is performed in this way, it will separate the digested peptide fragments in some manner apart from overall charge. Thus, when chromatography has completed, the digested peptides will have been separated first by overall charge, then by a property driven by the chromatography liquid, hence the “two-dimensional” separation.
The skilled artisan will recognize that the chromatography buffer will differ based upon the desired property for separation and will use that buffer that will give optimal separation of A the peptides in question. By way of example only, chromatography buffers may be selected that separate according to hydrophobicity, alkalinity, water solubility, or any other common means of separation apart from overall charge.
- Protein Chips
Once chromatography is complete, the thin layer plate is dried and the digested peptide fragments thus separated are detected using common means (such as detection of a radioactively-labeled antibody).
Protein chip technology provides a means of rapidly screening sample compounds for their ability to hybridize to PDE3 isoform proteins, peptides or subunits immobilized on a solid substrate. Specifically contemplated are protein array-based technologies such as those disclosed by Cheng et al. (U.S. Pat. No. 6,071,394), Zanzucchi et al. (U.S. Pat. No. 5,858,804) and Lee et al. (U.S. Pat. No. 5,948,627), each of which is incorporated herein by reference in their entirety. These techniques involve methods for analyzing large numbers of samples rapidly and accurately. The technology capitalizes on the binding properties of proteins or peptides to screen samples.
A protein chip or array consists of a solid substrate upon which an array of proteins or peptides have been attached. For screening, the chip or array is contacted with a sample containing one or more test compounds that may function as PDE3 inhibitors or activators. The degree of stringency of binding of test compound to peptides may be manipulated as desired by varying, for example, salt concentration, temperature, pH and detergent content of the medium. The chip or array is then scanned to determine which proteins or peptides have bound to a test compound.
The structure of a protein chip or array comprises: (1) an excitation source; (2) an array of probes; (3) a sampling element; (4) a detector; and (5) a signal amplification/treatment system. A chip may also include a support for immobilizing the probe.
In particular embodiments, a protein or peptide may be tagged or labeled with a substance that emits a detectable signal. The tagged or labeled species may be fluorescent, phosphorescent, or luminescent, or it may emit Raman energy or it may absorb energy. When the protein or peptide binds to a test compound, a signal is generated that is detected by the chip. The signal may then be processed in several ways, depending on the nature of the signal. In alternative embodiments, the test compounds may be labeled.
The proteins or peptides may be immobilized onto an integrated microchip that also supports a phototransducer and related detection circuitry. Alternatively, PDE3 proteins or peptides may be immobilized onto a membrane or filter that is then attached to the microchip or to the detector surface itself. The proteins or peptides may be directly or indirectly immobilized onto a transducer detection surface to ensure optimal contact and maximum detection. A variety of methods have been utilized to either permanently or removably attach proteins to a substrate. When immobilized onto a substrate, the proteins are stabilized and may be used repeatedly.
Exemplary substrates include nitrocellulose, nylon membrane or glass. Numerous other matrix materials may be used, including reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane) and photopolymers which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules (U.S. Pat. Nos. 5,405,766 and 5,986,076, each incorporated herein by reference).
Binding of proteins or peptides to a selected support may be accomplished by any of several means. For example, proteins may be bound to glass by first silanizing the glass surface, then activating with carbodiimide or glutaraldehyde. Alternative procedures may use reagents such as 3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane (APTS) linked via amino groups. With nitrocellulose membranes, the protein probes may be spotted onto the membranes.
Specific proteins or peptides may first be immobilized onto a membrane and then attached to a membrane in contact with a transducer detection surface. This method avoids binding the protein onto the transducer and may be desirable for large-scale production. Membranes particularly suitable for this application include nitrocellulose membrane (e.g., from BioRad, Hercules, Calif.) or polyvinylidene difluoride (PVDF) (BioRad, Hercules, Calif.) or nylon membrane (Zeta-Probe, BioRad) or polystyrene base substrates (DNA.BIND™ Costar, Cambridge, Mass.).
Certain embodiments of the present invention involve antibody production against one or more PDE3 isoforms. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, incorporated herein by reference).
Methods for generating polyclonal antibodies are well known in the art. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition and collecting antisera from that immunized animal. A wide range of animal species may be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin may also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition may be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes may be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. Later booster injections may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal may be bled and the serum isolated and stored, and/or the animal may be used to generate MAbs. For production of rabbit polyclonal antibodies, the animal may be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody or a peptide bound to a solid matrix.
Monoclonal antibodies (MAbs) may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified expressed protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
The methods for generating monoclonal antibodies (NAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (e.g., Goding, pp. 65-66, 1986). For example, where the immunized animal is a mouse, one may use P3-NS-1-Ag4-1, Sp2/0, P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPCL11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).
Viable, fused hybrids are differentiated from the parental, unfused cells by culturing in a selective medium. The selective medium generally contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. A preferred selection medium is HAT. The only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like. The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones may then be propagated indefinitely to provide MAbs.
- Immunoassay Methods
In accordance with the present invention, fragments of the monoclonal antibody of the invention may be obtained from the monoclonal antibody produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention may be synthesized using an automated peptide synthesizer.
Immunocomplex formation. In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying or otherwise generally detecting peptides of interest. The PDE3 proteins or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention may be employed to detect or purify the PDE3 proteins or peptides. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987).
In general, the immunobinding methods include obtaining a sample suspected of containing a protein, peptide or antibody, and contacting the sample with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. Patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. One may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the target protein, peptide or corresponding antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
The immunodetection methods of the present invention may be of utility in the diagnosis of various disease states. A biological or clinical sample suspected of containing either the target protein or peptide or corresponding antibody is used. In certain embodiments, samples from patients with cardiomyopathy and/or pulmonary hypertension may be immunoassayed to determine the type and abundance of different PDE3 isoforms present in one or more tissues. Targeted therapy directed towards PDE3 may utilize inhibitors and/or activators known to be selective or specific for one or more PDE3 isoforms that are detected in the patient's affected tissues.
Immunohistochemistry. The antibodies of the present invention may be used in conjunction with fresh-frozen or formalin-fixed, paraffin-embedded tissue blocks prepared by immunohistochemistry (IHC). Any IHC method well known in the art may be used, such as those described in Diagnostic Immunopathology, 2nd edition. edited by, Robert B. Colvin, Atul K. Bhan and Robert T. McCluskey. Raven Press, New York., 1995, (incorporated herein by reference).
ELISA. Certain immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
- Methods of Immobilization
In one exemplary ELISA, antibodies binding to the PDE3 proteins of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the PDE3 isoforms, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen may be detected. Detection is generally achieved by the addition of a second antibody specific for the target protein, linked to a detectable label. This type of ELISA is a simple “sandwich ELISA”. Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. The skilled artisan will realize that a variety of ELISA and other immunoassay techniques are known in the art, any of which may be performed within the scope of the present invention.
In various embodiments, the PDE3 proteins or peptides or anti-PDE3 antibodies of the present invention may be attached to a solid surface (“immobilized”). In a preferred embodiment, immobilization may occur by attachment to a solid surface, such as a magnetic, glass or plastic bead, a plastic microtiter plate or a glass slide.
Immobilization of proteins or peptides may be achieved by a variety of methods involving either non-covalent or covalent interactions between the immobilized protein or peptide and an anchor. In an exemplary embodiment, immobilization may be achieved by coating a solid surface with a cross-linkable group, such as an amino, carboxyl, sulfhydryl, alcohol or other group and attaching a protein or peptide using a cross-linking reagent.
- Nucleic Acids
Homobifunctional reagents that carry two identical functional groups are highly efficient in inducing cross-linking. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino or carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. Exemplary methods for cross-linking molecules are disclosed in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511. Amine residues may be introduced onto a surface through the use of aminosilane. Cross-linking reagents include bisimidates, dinitrobenzene, N-hydroxysuccinimide ester of suberic acid, disuccinimidyl tartarate, dimethyl-3,3′-dithiobispropionimidate, N-succinimidyl-3-(2-pyridyldithio)-propionate, 4-(bromoaminoethyl)-2-nitrophenylazide, 4-azidogyloxal and a water soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The present invention is not limiting as to the cross-linking agents that may be used.
The present invention also provides, in another embodiment, genes encoding PDE3. As discussed below, a “PDE3 gene” may contain a variety of different bases and yet still produce a corresponding polypeptide that is indistinguishable functionally, and in some cases structurally, from the genes disclosed herein. Other embodiments of the invention may concern nucleic acids (antisense RNAs, ribozymes) that can bind to and inhibit transcription and/or translation of one or more RNA species encoding a PDE3A isoform protein. The design and production of antisense RNAs, or cDNAs encoding antisense RNAs, are well known in the art and any such known method may be used in the practice of the present invention (e.g., U.S. Pat. Nos. 6,210,892; 6,248,724; 6,277,981; 6,300,492; 6,303,374; 6,310,047; 6,365,345). In certain embodiments, an antisense RNA may be targeted against a particular PDE3A isoform, for example by selecting a target sequence that is present in one PDE3A isoform mRNA but not in another. The term “nucleic acid” encompasses single-stranded, double-stranded, triple-stranded DNA and/or RNA of any type, as well as analogs of and chemically modified forms of DNA and/or RNA.
- Nucleic Acids Encoding PDE3
Any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid. Cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate, or enhance the catalytic activity, regulatory properties or subcellular localization of PDE3 isoforms.
Nucleic acids may contain an entire gene, a cDNA, or a domain of a PDE3 isoform that expresses catalytic activity, or any other fragment of the sequences set forth herein. The nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA).
- Assay of PDE3A Isoform mRAA Levels
The DNA segments of the present invention include those encoding biologically functional equivalent PDE3 proteins and peptides. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
Some embodiments of the invention concern methods for determining the levels of mRNA species encoding the three PDE3A isoforms in various cells, tissues, organs or other samples. A variety of assays for mRNA levels are known in the art and any such known assay may be used. The three PDE3A isoform mRNAs differ in length, not in sequence. Therefore, any assay for mRNA levels must either separate the mRNAs by size or must be performed by a subtraction process. The skilled artisan is aware that RNA species are particularly sensitive to endogenous and/or exogenous RNAse degradation and that great care must be taken to inhibit or inactivate RNAse before RNA levels can be determined. Typical procedures involve treatment of solutions with diethylpyrocarbonate (DEPC) and autoclaving as well as addition of commercial RNAse inhibitors.
Northern blotting is a well-known method for assaying mRNA species that differ by size. Either total cell RNA or polyadenylated mRNA may be purified from a sample by known techniques (e.g., Sambrook et al., 1989). The purified RNA is separated by size using gel electrophoresis. After transfer to a nylon, nitrocellulose or other membrane, the size separated RNAs are probed with a labeled oligonucleotide that hybridizes specifically with one or more target RNAs. The presence of an RNA species that hybridizes with the oligonucleotide probe is detected by autoradiography, fluorography or other known techniques. Further examples of the use of Northern blotting to detect PDE3A mRNAs are disclosed below in the Examples section. It appears that in most cell types, a given PDE3A isoform mRNA will either be present or absent. Thus, generally it will be sufficient to detect the presence or absence of a PDE3A isoform mRNA. However, the amounts of each isoform mRNA present in a sample may also be determined by standard techniques, such as using autoradiography or fluorography to expose a film (e.g. Kodak X-Omat, Eastman Kodak, Rochester, N.Y.), and scanning the band intensity on the developed film.
Other well known methods for detecting and/or quantifying mRNA species may be used. For example, the target nucleic acids of interest may be amplified as disclosed below. Amplification products may be attached to a membrane, 96-well plate, nucleic acid chip or other substrate and detected. Because the PDE3A isoforms do not differ in sequence, determination of the amounts of each mRNA species would require three separate probes. One probe would be designed to be complementary to the PDE3A3 mRNA sequence and would detect PDE3A1, PDE3A2 and PDE3A3. A second probe would be designed to be complementary to the 5′ portion of the PDE3A2 mRNA sequence (see SEQ ID NO: 15), for example to the 3′ end of exon 1 or to exons 2 or 3. That probe would hybridize with mRNAs for PDE3Al and PDE3A2. A third probe would be designed to be complementary with the 5′ end of exon 1. That probe would only hybridize with the mRNA encoding PDE3A1 (SEQ ID NO:14, SEQ ID NO:18). By assaying the levels of PDE3A mRNAs using the three different probes, it would be possible to determine the amount of each isoform mRNA species by subtraction.
As discussed in further detail in the Examples section, the PDE3A isoforms are encoded by at least two, and possibly by three different mRNAs. PDE3A1 mRNA is translated to a 136 kDa protein isoform, while a PDE3A2 mRNA may be translated to give both 94 kDA and 118 kDA protein isoforms. Alternatively, each of the different sized protein isoforms may be encoded by a separate mRNA species.
- High Through-Put Screening
Apparatus and kits for assay of mRNA expression levels are commercially available, such as the Nanochip™ Workstation (Nanogen, Inc., San Diego, Calif.), Affymetrix Genechip® (Affymetrix, Inc., Santa Clara, Calif.), etc.
In certain embodiments of the invention, high throughput screening (HTS) methods directed towards mRNA may be used to assay for inhibitors and/or activators that affect expression of specific PDE3 isoforms. Such methods are known in the art and in some embodiments may be performed using kits and/or apparatus obtained from commercial vendors (e.g. Xpress-Screen mRNA Detection Assay Service, Applied Biosystems, Foster City, Calif.). The object of high throughput screening is to survey thousands of compounds, for example in the form of small molecule libraries, phage display libraries, native plant or animal extracts, combinatorial chemistry libraries, etc. for a pharmaceutically significant effect on a target protein, cell, tissue, organ or organism. Effective compounds may be further modified by chemical substitution and/or modification to provide increased efficacy, safety, duration of effect, etc.
HTS assays may be directed against one or more proteins or peptides of interest, such as PDE3A-136, PDE3A-118, PDE3A-94 or PDE3B-137 using known techniques. Preferably, libraries of potential inhibitors and/or activators are exposed to PDE3 proteins and/or peptides and enzyme catalytic activity and/or regulatory properties are assayed. Such assays may be performed, for example, in 96-well microtiter plates using known colorimetric, luminescent and/or radioactive assays for enzyme activity. In other alternative embodiments, the test peptides and/or proteins may be attached to a surface, such as a protein chip, microtiter wells, membrane or other surface known in the art and libraries of compounds may be screened for their ability to bind to the various PDE3 isoforms.
Protein based HTS assays can be laborious and time-consuming. An alternative method for performing HTS analysis is to screen targets, such as cells, tissues, organs or organisms for an effect of a test compound on mRNA levels. With respect to PDE3 isoforms, such assays may potentially be directed towards identifying compounds that directly or indirectly affect PDE3A1 or PDE3A2 mRNA levels. The cell or tissue of interest, for example a tissue sample from an individual with dilated cardiomyopathy or an Sfo cell transfected with a PDE3A-encoding gene may be exposed to a series of test compounds in 96- or 384-well microplates. After incubation and cell lysis, a biotinylated probe specific for the mRNA of interest is used to hybridize to total cell RNA or to purified polyadenylated mRNA. The DNA-RNA hybrid may be transferred to a streptavidin coated plate, which binds to the biotinylated probe. A labeled antibody, such as an alkaline phosphatase conjugated antibody, that binds specifically to RNA-DNA hybrids is incubated with the plate, unbound antibody is removed by washing and the presence of RNA-DNA hybrids is detected by developing the labeled antibody, for example using a chemiluminescent substrate (Xpress-Screen, Applied Biosystems). In this way, hundreds of test compounds may be screened simultaneously for an effect on PDE3 isoform expression.
- Nucleic Acid Amplification
In alternative embodiments, test compounds may be screened by looking for secondary effects of PDE3A isoform proteins. Inhibition or activation of PDE3 activity and/or expression may be determined indirectly. By affecting the cellular levels of cAMP and/or cGMP, PDE3 isoforms may affect the expression of known cyclic nucleotide regulated genes. Cells or tissues that have been exposed to test compounds may be screened, as described above, for mRNAs encoded by genes that are known to be dependent on cyclic nucleotide levels. Effects of inhibitors and/or activators of PDE3 isoforms may be monitored by screening normal, diseased and/or transformed cells for changes in expression levels of cAMP or cGMP regulated genes.
Nucleic acids of use as a template for amplification may be isolated from cells contained in a biological sample, according to standard methodologies. (Sambrook et al., 1989) The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification. In other embodiments, the RNA may be polyadenylated mRNA. Purification of mRNA, for example by affinity chromatography to oligo-dT columns, is well known in the art.
Pairs of primers that selectively hybridize to nucleic acids corresponding to specific markers are contacted with the isolated nucleic acid under conditions that permit selective hybridization. Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced.
Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology, Bellus, 1994).
Following detection, one may compare the results seen in a given patient with a statistically significant reference group of normal patients and patients exhibiting a disease state. In this way, it is possible to correlate the amount of marker detected with various clinical states.
- Template Dependent Amplification Methods
The term primer, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences may be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each of which is incorporated herein by reference in its entirety.
A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reaction methodologies are well known in the art.
Another method for amplification is the ligase chain reaction (“LCR”), disclosed in European Application No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.
An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention. Walker et al., Proc. Nat'l Acad. Sci. USA 89:392-396 (1992), incorporated herein by reference in its entirety.
Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases may be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences may also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′ sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.
Still other amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, “modified” primers are used in a PCR like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR. Kwoh et al., Proc. Nat'l Acad. Sci. USA 86:1173 (1989); Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference in their entirety. In NASBA, the nucleic acids may be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.
Davey et al., European Application No. 329 822 (incorporated herein by reference in its entirely) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5′ to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence may be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies may then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification may be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence may be chosen to be in the form of either DNA or RNA.
Miller et al., PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include “race” and “one-sided PCR.” Frohman, M. A., In: PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y. (1990) and Ohara et al., Proc. Nat'l Acad. Sci. USA, 86:5673-5677 (1989), each herein incorporated by reference in their entirety.
- Separation Methods
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting “di-oligonucleotide”, thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention. Wu et al., Genomics 4:560 (1989), incorporated herein by reference in its entirety.
Following amplification, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al., 1989.
- Identification Methods
Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products may then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety.
In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art and may be found in many standard books on molecular protocols. See Sambrook et al., 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
- Antisense Constructs, Ribozymes and Small Interfering RNAs
One example of the foregoing is described in U.S. Pat. No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
The term “antisense” refers to polynucleotide molecules complementary to a portion of a targeted gene or mRNA species. Complementary polynucleotides are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, purines will base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
The intracellular concentration of monovalent cations is approximately 160 mM (10 mM Na+; 150 mM K+). The intracellular concentration of divalent cations is approximately 20 mM (18 mM Mg+; 2 mM Ca++). The intracellular protein concentration, which would serve to decrease the volume of hybridization and, therefore, increase the effective concentration of nucleic acid species, is 150 mg/ml. Constructs may be tested for specific hybridization in vitro under conditions that mimic these in vivo conditions.
Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. In certain embodiments, it is contemplated that effective antisense constructs may include regions complementary to the mRNA start site. In preferred embodiments, the antisense constructs are targeted to a sequence of an hnRNA and/or mRNA that is present in one PDE3A isoform and not in another. For example, one might target the 5′ end of the mRNA encoding PDE3A1 (SEQ ID NO:14, SEQ ID NO:18), which is missing the in the PDE3A2 mRNA (SEQ ID NO:15). One of ordinary skill in the art can readily test such constructs to determine whether levels of the target protein are affected.
As used herein, the terms “complementary” or “antisense” mean polynucleotides that are substantially complementary to the target sequence over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen nucleotides out of fifteen. Naturally, sequences that are “completely complementary” will be sequences that are entirely complementary throughout their entire length and have no base mismatches.
Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g., a ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
Although the antisense sequences may be full length cDNA copies, or large fragments thereof, they also may be shorter fragments, or “oligonucleotides,” defined herein as polynucleotides of 50 or less bases. Although shorter oligomers (8-20) are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of base-pairing. For example, both binding affinity and sequence specificity of an oligonucleotide to its complementary target increase with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 100 base pairs will be used. While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence of 14 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence.
In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).
Alternatively, the antisense oligo- and polynucleotides according to the present invention may be provided as RNA via transcription from expression constructs that carry nucleic acids encoding the oligo- or polynucleotides. Throughout this application, the term “expression construct” is meant to include any type of genetic construct containing a nucleic acid encoding a product in which part or all of the nucleic acid sequence is capable of being transcribed. Typical expression vectors include bacterial plasmids or phage, such as any of the pUC or Bluescript™ plasmid series or, as discussed further below, viral vectors adapted for use in eukaryotic cells.
In preferred embodiments, the nucleic acid encodes an antisense oligo- or polynucleotide under transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by an RNA polymerase to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation.
The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
The particular promoter that is employed to control the expression of a nucleic acid encoding the inhibitory polynucleotide is not believed to be important, so long as it is capable of expressing the peptide in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding the inhibitory peptide adjacent to and under the control of a promoter that is active in the human cell. Generally speaking, such a promoter might include either a human or viral promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level transcription. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art is contemplated as well, provided that the levels of transcription and/or translation are sufficient for a given purpose.
Selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of an antisense sequence. For example, a nucleic acid under control of the human PAI-1 promoter results in expression inducible by tumor necrosis factor. Additionally any promoter/enhancer combination also could be used to drive expression of a nucleic acid according to the present invention. Tables 1 and 2 list elements/promoters that may be employed to regulate transcription and/or translation of operably coupled genes. This list is exemplary only and any known promoter and/or regulatory element may be used.
|TABLE 1 |
|Immunoglobulin Heavy Chain |
|Immunoglobulin Light Chain |
|T-Cell Receptor |
|HLA DQ α and DQ β |
|Interleukin-2 Receptor |
|MHC Class II 5 |
|MHC Class II HLA-DRα |
|Prealbumin (Transthyretin) |
|Muscle Creatine Kinase |
|Elastase I |
|Albumin Gene |
|Neural Cell Adhesion Molecule (NCAM) |
|H2B (TH2B) Histone |
|Mouse or Type I Collagen |
|Glucose-Regulated Proteins (GRP94 and GRP78) |
|Rat Growth Hormone |
|Human Serum Amyloid A (SAA) |
|Troponin I (TN I) |
|Platelet-Derived Growth Factor |
|Duchenne Muscular Dystrophy |
|Papilloma Virus |
|Hepatitis B Virus |
|Human Immunodeficiency Virus |
|TABLE 2 |
|Element ||Inducer |
|MT II ||Phorbol Ester (TPA) |
| ||Heavy metals |
|MMTV (mouse mammary tumor ||Glucocorticoids |
|β-Interferon ||poly(rI)X, poly(rc) |
|Adenovirus 5 E2 ||Ela |
|c-jun ||Phorbol Ester (TPA), H2O2 |
|Collagenase ||Phorbol Ester (TPA) |
|Stromelysin ||Phorbol Ester (TPA), IL-1 |
|SV40 ||Phorbol Ester (TPA) |
|Murine MX Gene ||Interferon, Newcastle Disease Virus |
|GRP78 Gene ||A23187 |
|α-2-Macroglobulin ||IL-6 |
|Vimentin ||Serum |
|MHC Class I Gene H-2kB ||Interferon |
|HSP70 ||Ela, SV40 Large T Antigen |
|Proliferin ||Phorbol Ester-TPA |
|Tumor Necrosis Factor ||FMA |
|Thyroid Stimulating Hormone α ||Thyroid Hormone |
|Insulin E Box ||Glucose |
Another method for inhibiting the expression of specific PDE3A isoforms is via ribozymes. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., 1981). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to an internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., 1992). It was reported that ribozymes elicited genetic changes in some cells lines to which they were applied. The altered genes included the oncogenes H-ras, c-fos and genes of HIV.
Several different ribozyme motifs have been described with RNA cleavage activity (Symons, 1992). Examples that are expected to function equivalently include sequences from the Group I self splicing introns including Tobacco Ringspot Virus (Prody et al., 1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981), and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequences from these and related viruses are referred to as hammerhead ribozymes. Other suitable ribozymes include sequences from RNase P (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat. Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Delta virus based ribozymes (U.S. Pat. No. 5,625,047). The general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowrira et al., 1994).
The other variable in ribozyme design is the selection of a cleavage site on a given target RNA. Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence that is the cleavage site. For hammerhead ribozymes, the cleavage site is a dinucleotide sequence on the target RNA—a uracil (U) followed by either an adenine, cytosine or uracil (A,C or U) (Perriman et al., 1992).
- Small Interfering mRNAs
The large number of possible cleavage sites in genes of moderate size, coupled with the growing number of sequences with demonstrated catalytic RNA cleavage activity indicates that a large number of ribozymes that have the potential to downregulate gene expression are available. Additionally, due to the sequence variation among different genes, ribozymes could be designed to specifically cleave individual genes or gene products. Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al., (1994), incorporated by reference.
Another possibility is to inhibit the translation of individual PDE3 mRNAs by RNA interference. This method of post-transcriptional gene silencing involves the use of a 21- or 22-nucleotide double-stranded synthetic RNA molecule homologous to a unique nucleotide sequence in the mRNA of interest. Through a mechanism yet to be determined, such small interfering RNA molecules (siRNAs) have the ability to reduce expression of the cognate protein. This approach has been used to reduce the expression of several cytoskeletal proteins. As noted above, a unique sequence in PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) has been identified that may allow specific interference with the expression of PDE3A-136.
Methods for selectively interfering with gene expression using small interfering RNA species (“siRNA”) are known in the art (e.g., Bass, 2001; Elbashir et al., 2001). Short, double-stranded RNAs (dsRNA) of about 30 bp or less that are homologous in sequence to a gene to be silenced (e.g., PDE3A) are introduced into a target cell (Elbashir et al., 2001). By a poorly understood endogenous pathway, the dsRNAs are broken into smaller fragments of about 21-22 bp (siRNAs). These fragments trigger the degradation of homologous mRNA sequences (Elbashir et al., 2001), e.g. PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18). Use of siRNAs can decrease expression of a target gene or even eliminate it entirely (Bass, 2001). Another advantage of siRNAs is that they are effective at lower concentrations (about 1-25 nM) than antisense constructs (Bass, 2001; Elbashir et al., 2001).
- Expression Vectors
Transfection of 21 bp dsRNA sequences into NIH/3T3 cells, COS-7 cells and Hela S3 cells using cationic liposomes resulted in inhibition of homologous reporter genes (Elbashir et al., 2001). The effectiveness of inhibition appeared to be inversely related to the expression levels of the target gene, with highly expressed genes showing less inhibition by siRNA constructs (Elbashir et al., 2001). Because the PDE3 genes are expressed at relatively low levels compared to highly expressed mammalian genes, the use of siRNA inhibitors should prove effective at inhibiting or eliminating expression of targeted PDE3 isoforms.
Nucleic acids encoding PDE3 isoform proteins or peptides may be incorporated into expression vectors for production of the encoded proteins or peptides. Non-limiting examples of expression systems known in the art include bacteria such as E. coli, yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as in COS or CHO cells. A complete gene can be expressed or, alternatively, fragments of the gene encoding portions of polypeptide can be produced.
The gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques. An E. coli expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of the protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (1131, New Haven, Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.).
Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. Other fusion systems are designed to produce fusions wherein the fusion partner is easily excised from the desired polypeptide. In one embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).
The expression system used may also be one driven by the baculovirus polyhedron promoter. The gene encoding the polypeptide may be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. One baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant antigen. See U.S. Pat. No. 4,215,051 (incorporated by reference).
Amino acid sequence variants of the polypeptide may also be prepared. These may, for instance, be minor sequence variants of the polypeptide which arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants may be prepared by standard methods of site-directed mutagenesis such as those described herein.
Substitutional variants typically contain an alternative amino acid at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar size and charge. Conservative substitutions are well known in the art and include, for example, the changes of: arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine or glutamine; methionine to leucine or isoleucine; phenylalanine to tyrosine; serine to threonine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide. For example, an insertional variant may include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants may include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site.
The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of the claimed nucleic acid sequences.
As used herein, the terms “engineered” and “recombinant” cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene has been introduced through the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced exogenous DNA segment or gene. Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a heterologous promoter not naturally associated with the particular introduced gene.
To express a recombinant encoded protein or peptide, whether mutant or wild-type, in accordance with the present invention one would prepare an expression vector that comprises one of the claimed isolated nucleic acids under the control of, or operatively linked to, one or more promoters. To bring a coding sequence “under the control of” a promoter, one positions the 5′ end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides “downstream” (i.e., 3′) of the chosen promoter. The Express Mail No.: EV 118157766 US 51 “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in this context.
Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.
Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.
For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the trp1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
In addition to micro-organisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.
In a useful insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The isolated nucleic acid coding sequences are cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequences results in the inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the protein coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051 (Smith)).
Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the encoded protein.
Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems may be chosen to ensure the correct modification and processing of the foreign protein expressed. Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
The promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
A number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hind III site toward the Bg1 I site located in the viral origin of replication.
In cases where an adenovirus is used as an expression vector, the coding sequences may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing proteins in infected hosts.
Specific initiation signals may also be required for efficient translation of the claimed isolated nucleic acid coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons may be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators (Bittner et al., 1987).
In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides “downstream” of the termination site of the protein at a position prior to transcription termination.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding proteins may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells may be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn may be cloned and expanded into cell lines.
- Site-Specific Mutagenesis
A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962) and adenine phosphoribosyltransferase genes (Lowy et al., 1980), in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance may be used as the basis of selection for dhfr, that confers resistance to methotrexate (Wigler et al., 1980; O'Hare et al., 1981); gpt, that confers resistance to mycophenolic acid (Mulligan et al., 1981); neo, that confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981); and hygro, that confers resistance to hygromycin (Santerre et al., 1984).
Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13: phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
- Phage Display
In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector that includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
In certain embodiments, it may be desirable to use random amino acid sequences in the form of a phage display library for use as potential isoform selective PDE3 inhibitors or activators. The phage display method has been used for a variety of purposes (see, for example, Scott and Smith, 1990, 1993; U.S. Pat. Nos. 5,565,332, 5,596,079, 6,031,071 and 6,068,829, each incorporated herein by reference).
Generally, a phage display library is prepared by first constructing a partially randomized library of cDNA sequences, encoding a large number of amino acid combinations. The cDNA sequences are inserted in frame into, for example, a viral coat protein for a phage such as the fuse 5 vector (U.S. Pat. No. 6,068,829). The cDNAs are expressed as random amino acid sequences, incorporated into a coat protein. The randomized peptides are thus displayed on the external surface of the phage, where they can bind to proteins or peptides. Phage binding to PDE3 proteins or peptides may be separated from unbound phage using standard methods, for example by affinity chromatography to PDE3 peptides covalently linked to a solid support such as a membrane or chromatography beads. If desired, it is possible to collect bound phage, detach them from the PDE3 peptides by exposure to an appropriate solution and proceed with another round of binding and separation. This iterative process results in the selection of phage with an increased specificity for PDE3.
Once phage of an appropriate binding stringency have been obtained, it is possible to determine the amino acid sequence of the binding peptide by sequencing the portion of the phage genome containing the cDNA, for example by using PCR primers that flank the cDNA insertion site. Phage lacking any cDNA insert may be used as a control to ensure that binding is specific.
- Methods for Screening Active Compounds
The skilled artisan will realize that phage display may be used to select for peptides (between 3 and 100, more preferably between 5 and 50, more preferably between 7 and 25 amino acid residues long) that can bind to a desired protein or peptide. Such peptides may be of use, for example, as potential inhibitors or activators of PDE3 catalytic activity or protein-protein binding.
- In Vitro Assays
The present invention also contemplates the use of PDE3 isoform proteins, peptides and active fragments, and nucleic acids encoding PDE3, in the screening of potential PDE3 inhibitors or activators. These assays may make use of a variety of different formats and may depend on the kind of “activity” for which the screen is being conducted. Contemplated functional “read-outs” include binding to a substrate (e.g., cAMP or cGMP), inhibition of binding to a membrane or another protein, phosphorylation or dephosphorylation of PDE3, or inhibition or stimulation of a variety of cAMP dependent processes such as calcium channel activation or protein kinase activity.
In one embodiment, the invention is to be applied for the screening of compounds that bind to the PDE3 isoforms or a fragment thereof. The polypeptide or fragment may be either free in solution, fixed to a support, or expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting the determination of binding.
In another embodiment, the assay may measure the inhibition of binding of PDE3 to a natural or artificial substrate or binding partner. Competitive binding assays can be performed in which one of the agents is labeled. Usually, the polypeptide will be the labeled species. One may measure the amount of free label versus bound label to determine binding, or inhibition of binding.
Another technique for high throughput screening of compounds is described in WO 84/03564, the contents of which are incorporated herein by reference. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with PDE3 and washed. Bound polypeptide is detected by various methods.
Purified PDE3 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase. Also, fusion proteins containing a reactive region (preferably a terminal region) may be used to link the PDE3 active region to a solid phase.
- In Vivo Assays
Various cell lines containing wild-type or natural or engineered mutations in PDE3 can be used to study various functional attributes of these proteins and how a candidate compound affects these attributes. Methods for engineering mutations are described elsewhere in this document. In such assays, the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell. Depending on the assay, culture may be required. The cell may then be examined by virtue of a number of different physiologic assays. Alternatively, molecular analysis may be performed in which the function of PDE3 or related pathways, may be explored. This may involve assays such as those for phosphorylation states of various molecules, cAMP levels, mRNA expression for CREB linked genes, or any other process regulated in whole or in part by PDE3 activity. For certain embodiments, it may be desirable to create “knock-out” cells that are lacking in endogenous phosphodiesterase activity in order to specifically assay the effects of various compounds on inserted isoforms of PDE3.
The present invention also encompasses the use of various animal models. By developing or isolating mutant cells lines that show differential expression of one or more PDE3 isoforms, one can generate animal models that will be predictive of cardiomyopathy and/or pulmonary hypertension in humans and other mammals. These models may employ transgenic animals that differentially express one or more PDE3 isoforms.
Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous or intraarterial injection.
- Rational Drug Design
Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to: survival, increased cardiac output; increased ventricular ejection fraction; reduced pulmonary arterial pressure; improved exercise tolerance; improved quality-of-life index; reduced incidence of myocardial ischemia or infarction; reduced incidence of ventricular ectopic activity or arrhythmia; reduced or increased blood pressure; decreased myocardial mass (reduced hypertrophy); reduced vascular hyperplasia; reduced vascular resistance; reduced platelet aggregation.
The goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs that are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for PDE3 or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling based on the 3-D structures of other phosphodiesterases or by a combination of both approaches. In addition, knowledge of the polypeptide sequences permits computer employed predictions of structure-function relationships. An alternative approach, an “alanine scan,” involves the random replacement of residues throughout a protein or peptide molecule with alanine, followed by determining the resulting effect(s) on protein function.
It also is possible to isolate a PDE3 specific antibody, selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of an anti-idiotype antibody would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
Thus, one may design drugs that have improved PDE3 isoform selective activity or which act as stimulators, inhibitors, agonists, or antagonists of PDE3.
The technique known as homologous recombination allows the precise modification of existing genes, including the inactivation of specific genes, as well as the replacement of one gene for another. Methods for homologous recombination are described in U. S. Pat. No. 5,614,396, incorporated herein by reference.
Homologous recombination relies on the tendency of nucleic acids to base pair with complementary sequences. In this instance, the base pairing serves to facilitate the interaction of two separate nucleic acid molecules so that strand breakage and repair can take place. In other words, the “homologous” aspect of the method relies on sequence homology to bring two complementary sequences into close proximity, while the “recombination” aspect provides for one complementary sequence to replace the other by virtue of the breaking of certain bonds and the formation of others.
First, a site for integration is selected within the host cell, such as the PDE3A or PDE3B genes. Sequences homologous to the integration site are included in a genetic construct, flanking the selected gene to be integrated into the genome. Flanking, in this context, simply means that target homologous sequences are located both upstream (5′) and downstream (3′) of the selected gene. The construct is then introduced into the cell, permitting recombination between the cellular sequences and the construct.
It is common to include within the construct a selectable marker gene. This gene permits selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic and biocidal drugs. In addition, this technique may be used to “knock-out” (delete) or interrupt a particular gene. Thus, another approach for inhibiting gene expression involves the use of homologous recombination, or “knock-out technology”. This is accomplished by including a mutated or vastly deleted form of the heterologous gene between the flanking regions within the construct. The arrangement of a construct to effect homologous recombination might be as follows:
vector•5′-flanking sequence•selected gene•selectable marker gene•flanking sequence-3′•vector
Using this kind of construct, it is possible, in a single recombinatorial event, to (i) “knock out” an endogenous gene, (ii) provide a selectable marker for identifying such an event or (iii) introduce a transgene for expression.
- Formulations and Routes for Administration to Patients
Another refinement of the homologous recombination approach involves the use of a “negative” selectable marker. One example of the use of the cytosine deaminase gene in a negative selection method is described in U.S. Pat. No. 5,624,830. The negative selection marker, unlike the selectable marker, causes death of cells that express the marker. Thus, it is used to identify undesirable recombination events. When seeking to select homologous recombinants using a selectable marker, it is difficult in the initial screening step to identify proper homologous recombinants from recombinants generated from random, non-sequence specific events. These recombinants also may contain the selectable marker gene and may express the heterologous protein of interest, but will, in all likelihood, not have the desired phenotype. By attaching a negative selectable marker to the construct, but outside of the flanking regions, one can select against many random recombination events that will incorporate the negative selectable marker. Homologous recombination should not introduce the negative selectable marker, as it is outside of the flanking sequences.
In certain embodiments, the isoform-selective inhibitors or activators of PDE3 may be used for therapeutic treatment of medical conditions, such as dilated cardiomyopathy and/or pulmonary hypertension. Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
Aqueous compositions of the present invention comprise an effective amount of PDE3 inhibitor or activator, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as innocula. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the PDE3 inhibitors or activators of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions.
The active compounds also may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts which are formed by reaction of basic groups with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free acidic groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
- Example 1
Materials and Methods
Preparation of rtPDE3A1
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
A human myocardial PDE3A construct was generated by inserting an eight amino acid Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) immediately upstream from the stop codon of PDE3A1 (SEQ ID NO:14). Using 50 ng PDE3A1 cDNA as template (GenBank accession number NM—
000921), PCR amplification was performed in a GeneAmp PCR system (Perkin Elmer, Wellesley, Mass.) with Pfu polymerase (Stratagene, La Jolla, Calif.) using 3 pmol each of sense primer corresponding to nt 3009-3027 of the PDE3A1 ORF CTTCATCTCTCACATTGTGGGGCCTCTGTG (SEQ ID NO:4) and antisense primer corresponding to nt 3423-3403 and the Flag epitope.
|(SEQ ID NO:5) || |
|TTTGCGGCCGCCTCGAGTTATTTATCATCATCATCTTTATAATCCTGGT || |
The resulting PCR product contained a unique PDE3 DraIII site at the 5′ end and a stop codon at the 3′ end. The stop codon was flanked upstream by a Flag epitope-coding sequence and downstream by an XhoI site. The PCR products were subcloned into the pCRII vector (Invitrogen, Carlsbad, Calif.) and isolated from this vector as DraIII/XhoI fragments. XhoI/DraIII fragments containing the ORF sequence of PDE3A1 (SEQ ID NO:14) upstream from the unique DraIII site were restricted from pBluescript. In a three-way ligation, these 5′ XhoI/DraIII fragments were ligated via the DraIII site to the 3′ DraIII/XhoI Flag epitope-containing fragments and to XhoI-cut pZero vector (Invitrogen), to give PDE3A1 Flag-pZero. PDE3A1-Flag was then excised from pZero with XhoI, ligated into pAcSG2 vector, subcloned and amplified.
- Preparation of Subcellular Fractions of Human Myocardium and Cultured Aortic Myocytes
PDE3A1-Flag-pAcSG2 plasmid (2 μg) was co-transfected with linearized BaculoGold DNA into Sf21 cells (BaculoGold transfection kit; Pharmingen, San Diego, Calif.). After five days, fresh Sf21 cells, (10-20)×106 cells per 75 cm2 flask, grown in TNM-FH media (BD-Pharmingen, San Diego, Calif.), were infected with medium containing PDE3A1-Flag baculovirus. For amplification, 100-500 μl of medium was collected after 72-96 h and used to infect fresh cultures, after which viral titers were determined by twelve-well end-point dilution assay. Cells from 75cm2 flasks, usually 10-20×106 cells per flask, were sedimented for 10 min at 1000×g, washed twice with ice-cold PBS and resuspended in 10 mM HEPES, 1 mM EDTA, 250 mM sucrose, 10 mM pyrophosphate, 5 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 1% NP-40 and 10 μg/ml each of aprotinin, leupeptin and pepstatin. Lysates were prepared by sonication on ice (two 20-second pulses, output 2, 40% of cycle) with a Sonifier Cell Disruptor 350 (Branson Sonic Power, Danbury, Conn.). Lysates were sedimented for 10 min at 12,000×g; supernatant fractions were used for Western blotting. C-terminally Flag-tagged rtPDE3A1 was purified to apparent homogeneity by immunoprecipitation with anti-Flag antibodies followed by competitive release with Flag peptide.
- Western Blotting
Cytosolic and KC1-washed microsomal fractions, from the left ventricular myocardium of explanted hearts of cardiac transplant recipients with idiopathic dilated cardiomyopathy, were prepared by homogenization, differential sedimentation and high-salt washing. Each preparation was made from tissue pooled from at least three different hearts. Tissue from left ventricular free walls was trimmed of epicardium and endocardium, cut into roughly 0.5 cm3 pieces, rapidly frozen in liquid nitrogen, and stored at −80° C. until use. To prepare subcellular fractions, 0.3 g of the frozen tissue were added to 5 volumes of buffer (5 mM KH2PO4/K2HPO4 and 2 mM EDTA (pH 6.8, 4° C.), 1 mM dithiothreitol, 1 mM benzamidine, 0.8 mM PMSF, and 1 μg/ml each of pepstatin A, leupeptin, and antipain). The tissue was homogenized twice for 10 seconds each. The homogenate was sedimented at 14,000 rpm for 20 minutes using an Eppendorf Model 5415 centrifuge. The supernatant was saved and the pellet resuspended in 1.5 ml of buffer, then rehomogenized and resedimented in order to solubilize any trapped cytosolic proteins. The supernatants containing cytosolic proteins were pooled and diluted 1:1 with buffer containing 40% v/v glycerol and stored at −80° C. until use. Comparable fractions of cultured human aortic myocytes (Clonetics, East Rutherford, N.J.; seventh passage) were similarly prepared.
- Expression of rtPDE3A1 Isoforms by in vitro Transcription/translation
Lysates of Sf21 cells expressing rtPDE3A1 and subcellular fractions of human myocardium and aortic myocytes were precipitated with trichloroacetic acid (final concentration 50%), dissolved in SDS buffer, subjected to SDS-PAGE (8% acrylamide) and transferred electrophoretically to nitrocellulose membranes (Schleicher & Schuell, Kenne, N.H.). After transfer, membranes were blocked, washed and incubated for at least 2 h at room temperature with polyclonal antibodies raised against synthetic peptides whose sequences correspond to selected regions of the open reading frame of PDE3A1 (SEQ ID NO: 14). The polyclonal antibodies corresponded to N-terminal amino acids 29-42 (anti-NT), mid-sequence amino acids 424-460 (anti-MID), and C-terminal amino acids 1125-1141 (anti-CT) of PDE3A1 (see SEQ ID NO:1). Immunoreactive bands were detected with a horseradish peroxidase-conjugated second antibody (Promega, Madison, Wis.) and an enhanced chemiluminescence luminescent reagent (Pierce, Rockford, Ill.) in accordance with the manufacturer's instructions.
The entire coding region of PDE3A1 cDNA (SEQ ID NO:14) was inserted into pBluescript. In addition, a plasmid with an ATGATG to CTGCTG mutation (Met-Met>Leu-Leu) at nt 1450-5 (ATG7/8) was generated by PCR using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.). The sense primer GGAATAATCCAGTGCTGCTGACCCTCACCAAAAGCAGATCC (SEQ ID NO:6) and the complementary anti-sense primer (corresponding to nt 1435-76 of the PDE3A1 ORF-SEQ ID NO:14) were used for mutagenesis. After amplification in E. coli (XL1-Blue), mutated plasmids were purified using a QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.) and sequenced.
PCR products with different 5 deletions were generated from the wild-type and mutated pBluescript-PDE3A1 plasmids using five sense primers containing T7 promoter sites immediately upstream from gene-specific sequences and an anti-sense primer containing the stop codon and a poly-A tail. The sense primers used for amplification in these reactions were as follows.
|(SEQ ID NO:7) || |
|TAATACGACTCACTATAGGGAGTGAAGAGGGCACCCTATACCATGGCAG || |
|(SEQ ID NO:8) || |
|TAATACGACTCACTATAGGGTTCAGTCTCCTGTGTGCCTTCTTCTGGATG || |
|(SEQ ID NO:9) || |
|TAATACGACTCACTATAGGGGAAGCGCTCGTCCAGATTGGGCTGGGC || |
|(SEQ ID NO:10) || |
|TAATACGACTCACTATAGGGTGGAGACCTTACCTGGCGTACCTGGCC || |
|(SEQ ID NO:11) || |
|TAATACGACTCACTATAGGGACTGCAGGAAGCACCTTCATCCAGTCC || |
The primers correspond, respectively, to nucleotides (−)22 to (−)7 in the 5-untranslated region, nucleotides 409-438, nucleotides 511-537, nucleotides 706-732, and nucleotides 1401-1427 of the open reading frame of PDE3A1 (SEQ ID NO:14, GenBank Accession No. NM000921). In each case, the antisense primer, corresponding to nucleotides 3400-3426 of PDE3A1 (SEQ ID NO: 14), was TTTTTTTTTTTTTTTTTTTTTCACTGGTCTGGCTTTTGGGTTGGTAT (SEQ ID NO: 12)
- 5′ RACE
In vitro translation products were synthesized from the PCR fragment templates and labeled with 4 μCi [35S]methionine (1000 Ci/mmol) in reticulocyte lysates using the TnT T7 Quick for PCR DNA system (Promega, Madison, Wis.). To make a synthetic protein, a PCR product containing a 5 deletion and a T7 promoter sequence was added to the TnT T7 PCR Quick Master Mix and incubated for roughly 60-90 minutes at 30° C. This process was repeated for each PCR construct containing a 5 deletion. Proteins thus created were isolated and subsequently analyzed by autoradiography.
- Southern and Northern Blotting
PCR amplification was performed on Marathon RACE-Ready cDNA from human myocardium (Clontech, Palo Alto, Calif.) using 1 pmol gene-specific anti-sense primer and 1 pmol sense primer corresponding to the 5′ end of the manufacturer's 5′ tag. A second round of PCR was performed for 35 cycles using 1 pmol nested gene-specific primer and 1 pmol nested sense primer corresponding to a second sequence within the manufacturer's tag. RACE products were purified on agarose gels and ligated into the pCR2.1 vector with T4 ligase (14° C., overnight) using a TA cloning kit (Invitrogen, San Diego, Calif.). Competent cells (INV F′) were transformed using a One Shot Kit (Invitrogen, Austin, Tex.) and plated on X-ga1 LB-ampicillin plates (100 μg/ml ampicillin). Positive colonies were grown overnight in LB-ampicillin medium. Plasmids were purified using Mini- or Midiprep Plasmid purification systems (Qiagen) and inserts were excised with EcoRI. Insert sizes were estimated by electrophoresis through agarose gels.
- Example 2
PDE3 Isoforms in Cardiac and Vascular Myocytes
DNA probes were prepared from PDE3A1 plasmid by PCR using region-specific primers. PCR products were purified using QIA Quick Kits (Qiagen). DNA was labeled with [32P]dCTP (3000 Ci/mmol, 10 mCi/ml) using a random primer labeling kit (Stratagene). Unincorporated nucleotides were removed using Sepahadex G-50 (fine) columns (Roche, Indianapolis, Ind.). For Southern blotting, linear DNA corresponding to nt (−)268 to nt 2610 of the PDE3A1 ORF (SEQ ID NO:14, SEQ ID NO:18) was prepared from PDE3A1 template by PCR and purified as described above. The PCR product was quantified by measurement of the A260/A280 ratio and its purity confirmed by agarose gel electrophoresis. PCR product samples were subjected to electrophoresis on 0.7% agarose gels, transferred to Gene Screen Plus Nylon Membranes (New England Nuclear, Boston, Mass.), crosslinked and pre-hybridized for 2-3 h in QuikHyb (Stratagene). Labeled DNA probes were hybridized with DNA blots at 65° C. for three to four hours using 1.25×106 cpm/ml of probe and 0.1 mg/ml salmon sperm DNA. Following hybridization, excess radiolabeled probe was removed by rinsing in SSC/0.1% SDS and autoradiography was performed at −80° C. For Northern blotting, RNA was extracted from human left ventricular myocardium from the excised hearts of transplant recipients with dilated cardiomyopathy using TRI reagent (Molecular Research Center, Cincinnati, Ohio). PolyA RNA was prepared from total RNA using a Message Maker kit (Life Technologies, Rockville, Md.). RNA was quantified and its purity confirmed as described above. PolyA RNA samples were subjected to electrophoresis on 1% agarose 0.5 M formaldehyde gels, transferred to Gene Screen Plus Nylon Membranes, crosslinked and pre-hybridized in for 2-3 h in QuikHyb. Labeled DNA probes were hybridized with RNA blots, excess radiolabeled probe was removed
It has been shown that proteins of three different apparent molecular weights can be immunoprecipitated from mammalian myocardium with anti-PDE3 antibodies (Smith et al., 1993). These proteins are identified herein as PDE3 isoforms by Western blotting of cytosolic and microsomal fractions of human myocardium, using antibodies raised against peptides derived from the PDE3A ORF.
An antibody against the C-terminus of PDE3 (“anti-CT”) reacted with three proteins in these fractions (FIG. 5). The largest, with an apparent MW of 136,000 on SDS-PAGE (“PDE3A-136”), was present exclusively in microsomal fractions (FIG. 5). Another PDE3 isoform, with an apparent MW of 118,000 (“PDE3A-118”), was present in both microsomal and cytosolic fractions, as was a third isoform with an apparent MW of 94,000 (“PDE3A-94”) (FIG. 5).
An antibody against an amino acid sequence between NHR2 and CCR (“anti-MID”) reacted with PDE3A-136 and PDE3A-118 but not PDE3A-94 (FIG. 5). An antibody against amino acids 25-49 (“anti-NT”) did not react with any protein in microsomal or cytosolic fractions, indicating the absence of this region from cardiac and vascular PDE3 isoforms (FIG. 5). However, anti-NT did react with an rtPDE3A1 containing the full-length ORF (FIG. 5).
The antibodies were used to identify PDE3 isoforms in subcellular fractions of aortic myocytes (Choi et al., 2001). Anti-CT reacted with 94-kDA and 118-kDa proteins in microsomal and cytosolic fractions of aortic myocytes (not shown). Anti-MID reacted only with the 118-kDa proteins (not shown). No proteins were visualized with anti-NT, and the 136-kDa protein band was absent in all cases (not shown).
Western blotting was used to show that PDE3B is present in vascular myocytes, where it appears as a 137-kDa band in the microsomal fraction (PDE3B-137) (Liu and Maurice, 1998). Western blots (not shown) indicate PDE3B-137 is absent from myocardium (not shown). These results are summarized in Table 3.
|TABLE 3 |
|Distribution of PDE3 isoforms in cardiac and vascular myocytes |
| ||Isoform |
| ||PDE3A- ||PDE3A- ||PDE3A- ||PDE3B- |
|Cell/tissue ||Fraction ||136 ||118 ||94 ||137 |
|Cardiac ||Microsomes ||+ ||+ ||+ || |
| ||Cytosol || ||+ ||+ |
|Vascular ||Microsomes || ||+ ||+ ||+ |
| ||Cytosol || ||+ ||+ |
- Example 3
Mechanisms for Generating Cardiac and Vascular PDE3A Isoforms
All three polyclonal antibodies (anti-NT, anti-MID and anti-CT) reacted with recombinant PDE3A1. Anti-CT reacted with proteins in the cytosolic and microsomal fractions of human myocardium that had apparent molecular weights of 94,000 Da and 118,000 Da. Anti-CT also reacted with a protein with an apparent molecular weight of 136,000 Da that was seen only in microsomal myocardial fractions. Anti-MID also reacted with the 118,000 and 136,000 proteins, but not the 94,000 Da protein.
Addition of [35S]-labeled rtPDE3A (full-length ORF, SEQ ID NO:14) to a sample of human myocardium prior to the preparation of cytosolic and microsomal fractions provided no evidence for the generation of smaller isoforms by proteolysis of the labeled full-length rtPDE3A (not shown). Other potential mechanisms were investigated.
The migration of cardiac and vascular isoforms of PDE3A were compared to those of recombinant proteins generated by in vitro transcription/translation. PDE3A constructs were prepared with 5′ deletions designed to yield rtPDE3A's starting from different in-frame ATG's, inserted downstream from a T7 promoter and Kozak sequence (FIG. 3). PDE3A-136, PDE3A-118 and PDE3A-94 migrated with the same apparent molecular weights as the rtPDE3A's starting at ATG's 1507, 1969 and 2521, respectively (FIG. 6). This is consistent with the three PDE3A isoforms being generated by transcription from alternative start sites. Transcription/translation from every PDE3A-derived construct generated an rtPDE3A whose apparent MW corresponded to PDE3A-94. To determine whether the latter might be generated by translation from a downstream AUG, a full-length rtPDE3A construct was prepared in which the ATG at nt 2521 was mutated to CTG (M to L). This mutation resulted in the disappearance of rtPDE3A-94 (not shown). It is concluded that the PDE3A-94 isoform is generated by transcription from the ATG initiation codon at nt 2521.
- Example 4
Structure-function Relationships in PDE3A Isoforms
At least two different messenger RNA species are expressed in different tissues—PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) in cardiac myocytes and PDE3A2 mRNA (SEQ ID NO:15) in both cardiac and vascular myocytes (Choi et al., 2001). It appears that transcription from alternative start sites in PDE3A results in the expression of PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) in cardiac myocytes and of PDE3A2 mRNA (SEQ ID NO:15) in cardiac and vascular myocytes. From the above results it is concluded that PDE3A-136 is generated in cardiac myocytes by translation from the second AUG in PDE3A1 mRNA (SEQ ID NO:14), while PDE3A-118 and PDE3A-94 are generated in cardiac and vascular myocytes by translation from alternative downstream AUG's in PDE3A2 mRNA (SEQ ID NO:15) (FIG. 7).
- Example 5
Functional Domains of PDE3A Isoforms
FIG. 11 shows the complete amino acid sequence of the open reading frame (ORF) for PDE3A. To date, three isoforms of PDE3A have been characterized. These are apparently generated by N-terminal truncation of the PDE3A ORF (SEQ ID NO:14). The apparent N-terminal methionine residues of the three isoforms are indicated in bold in FIG. 11. Those are located at residues 146 for PDE3A-136, 300 for PDE3A-118 and either 484 or 485 for PDE3A-94. The locations of the phosphorylation sites on the PDE3A isoforms are indicated by underlining in FIG. 11. The P1 site is located at residues 288-294, the P2 site at residues 309-312 and the P3 site at residues 435-438. The P2 and P3 sites on the PDE3A isoforms contain a single serine residue and the phosphorylated amino acid is unambiguous. The P1 site contains multiple serine residues and it is presently unknown which of these is covalently modified by phosphorylation.
The functional domains in the cardiac and vascular isoforms of PDE3 are shown in Table 4. The domains were elucidated in part by comparison of the electrophoretic migration, via SDS-PAGE, of native PDE3 isoforms and recombinant PDE3A isoforms generated by in vitro transcription/translation from constructs with 5 deletions of the open reading frame designed to result in translation from different in-frame start codons (ATG codon sequences). The rtPDE3A deletion constructs and the locations of the different ATG start codons in the PDE3A1 ORF (SEQ ID NO:14) are illustrated in FIG. 12. All recombinant isoforms migrated with apparent molecular weights approximately 20,000 higher then predicted by their amino acid sequences (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3).
|TABLE 4 |
|Functional domains in cardiac and vascular PDE3 isoforms. |
| ||PK-B ||PK-A site || ||PK-A site, || |
| ||site ||upstream || ||downstream || |
| ||NHR1 ||(‘P1’) ||(‘P2’) ||NHR2 ||(‘P3’) ||CCR |
| || |
|PDE3B-137 ||+ ||+ ||+ ||+ ||+ ||+ |
|PDE3A-136 ||+ ||+ ||+ ||+ ||+ ||+ |
|PDE3A-118 || || ||+ ||+ ||+ ||+ |
|PDE3A-94 || || || || || ||+ |
The apparent molecular weight of PDE3A-136 was slightly higher than the apparent molecular weight of 131,000 for the recombinant protein as translated from ATG-2 in the PDE3A1 open reading frame (SEQ ID NO:14), indicating that PDE3A1-136 contains part of the NHR1 site (a finding consistent with its recovery only in microsomal fractions), all of NHR2, and the P1, P2 and P3 sites for phosphorylation and activation by PK-B and PK-A. PDE3A-136 is therefore generated in cardiac myocytes from PDE3A1 either by translation from ATG1 followed by targeted N-terminal proteolysis or by some post-translational modification that reduces its electrophoretic mobility, resulting in a higher apparent molecular weight.
The apparent molecular weight of PDE3A-118 was indistinguishable from that of the recombinant PDE3A translated from ATG4, indicating that PDE3A-118 lacks NHR1 and the PK-B activation site, but includes the NHR2 and PK-A sites. PDE3A-118 is generated in cardiac and vascular myocytes from PDE3A2 mRNA (SEQ ID NO:15) by translation from ATG4, the third ATG in the open reading frame predicted by the cloned cDNA (GenBank Accession No. NM000921), or by translation from ATG2 or ATG3 followed by targeted N-terminal proteolysis.
The apparent molecular weight of PDE3A-94 was approximately equal to the apparent molecular weight of 94,000 for the recombinant PDE3A translated from ATG7/8, indicating that PDE3A-94 contains neither the membrane-association domains NHR1 and NHR2, nor any of the three phosphorylation sites. PDE3A-94 is generated in cardiac and vascular myocytes from PDE3A2 mRNA (SEQ ID NO:15) either by translation from AUG7/8 or by translation from a more upstream ATG followed by proteolytic removal of a more extensive length of N-terminal sequence. That PDE3A-118 and PDE3A-94 are generated from a single mRNA (SEQ ID NO:15) by alternative translational processing in vivo is consistent with the observation that a PDE3A-94-like protein is generated from longer constructs by translation from downstream ATG sequences in vitro.
The N-terminus predicted from the PDE3A1 open reading frame (SEQ ID NO:14) was absent from native PDE3A-136, the longest PDE isoform identified. All three isoforms contain the same C-terminal amino acid sequences, downstream of different N-terminal starting points.
PDE3A-136 and PDE3B-137, which contain the transmembrane helices of NHR1, would be expected to be exclusively membrane-bound in cardiac and vascular myocytes. PDE3A-118, which contains NHR2 but not NHR1, and PDE3A-94, which lacks both NHR1 and NHR2, would be expected to associate reversibly with intracellular membrane proteins or to be partitioned between the cytosolic and microsomal compartments. Their presence in both microsomal and cytosolic fractions is compatible with this conclusion. Further, the fact that PDE3A-118 and PDE3A-94 can be recovered in microsomal fractions suggests that interactions with anchoring or targeting proteins are involved in their intracellular localization.
The N-terminal sequence differences may cause different PDE3 isoforms to interact with different anchoring or targeting proteins that localize them to different signaling modules. As a consequence, each PDE3 isoform may regulate the phosphorylation of different substrates of PK-A and PK-G.
- Example 6
5 RACE PCR
Surprisingly, transcription/translation from every PDE3A-derived construct generated a recombinant PDE3A isoform whose apparent molecular weight corresponded to that of PDE3A-94. Determination whether the latter might be generated by translation from a downstream AUG in the full-length PDE3A mRNA (SEQ ID NO:14, SEQ ID NO:18) was performed by expression of a mutated construct starting from ATG1 in the PDE3A1 mRNA (SEQ ID NO:14) in which ATG7/8 was mutated to CTGCTG (Met-Met Leu-Leu). Expression of the mutated construct resulted in the disappearance of the 94,000 molecular weight recombinant PDE3A, a result consistent with the generation of PDE3A-94 from the full-length PDE3 mRNA by translation from AUG7/8.
Studies have shown that a PDE3A2 mRNA (SEQ ID NO:15), whose sequence is identical to that of the PDE3A1 cDNA downstream of roughly nucleotide 300 in the latter's open reading frame (SEQ ID NO:14) but which lacks PDE3A1's upstream sequence (SEQ ID NO:14, SEQ ID NO:18), is present in both cardiac and vascular myocytes, while PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) is present in cardiac but not in vascular myocytes (Choi, YH, et al., Biochem J., 2001). To determine if the PDE3A2 mRNA (SEQ ID NO:15) contained an alternative sequence upstream of roughly nucleotide 300, 5 RACE PCR was performed on a human myocardial cDNA library using three pairs of anti-sense primers derived from the shared sequences of PDE3A1 (SEQ ID NO:14) and PDE3A2 (SEQ ID NO:15).
- Example 7
Southern and Northern Blotting
Subcloning and sequencing of the multiple 5 RACE products indicated that the PDE3A2 mRNA (SEQ ID NO:15) contained no alternative sequence upstream of roughly nucleotide 300 (not shown). Similar results were obtained when 5 RACE was performed with comparable primers on a human aortic cDNA library (not shown).
Northern and Southern blotting was performed on nucleic acids from human left ventricular myocardium using probes derived from different regions of the PDE3A1 open reading frame (see SEQ ID NO: 14). The first nucleic acid probe, derived from nucleotides (−)268-189, corresponded to a region predicted to be present in PDE3A1 (SEQ ID NO:14), but not in PDE3A2 (SEQ ID NO:15). The other two nucleic acid probes used corresponded respectively to nucleotides 517-957 and 2248-2610 of PDE3A1 (SEQ ID NO:14), regions predicted to be present in both PDE3A1 (SEQ ID NO:14) and PDE3A2 (SEQ ID NO:15).
- Example 8
Inhibitors of PDE3 Activity and Effects of Intracellular Localization on Catalytic Activity
All three of the nucleic acid probes bound to an 8.2 kilobase band (not shown). The two downstream probes also bound to a 6.9 kilobase band to which the upstream probe did not bind (not shown). These results indicate that the 8.2 kilobase band is PDE3A1 (SEQ ID NO:14, SEQ ID NO:18) and the 6.9 kilobase band is PDE3A2 (SEQ ID NO:15). The size differences observed between the two hybridized bands are accounted for by the absence of the first roughly 300 nucleotides of the open reading frame of PDE3A1 (SEQ ID NO: 14) from PDE3A (SEQ ID NO:15), consistent with the generation of the latter by alternative transcription or splicing within exon 1 of the open reading frame of PDE3A1 (SEQ ID NO:14). This result is consistent with a result predicted by ribonuclease protection assays of RNA prepared from human myocardium and cultured human aortic myocytes with antisense probes spanning nucleotides 208-537 and nucleotides 2248-2610 of PDE3A1 (SEQ ID NO:14) (Choi, YH, et al., Biochem J., 2001). Importantly, PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) and PDE3A-136 were determined to be present in only cardiac myocytes while PDE3A2 mRNA (SEQ ID NO:15), PDE3A-118, and PDE3A-94 were present in both cardiac and vascular myocytes. This result indicates that PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) gives rise to PDE3A-136 and PDE3A2 mRNA (SEQ ID NO:15) gives rise to both PDE3A-118 and PDE3A-94.
The effects of two PDE3 inhibitors, cilostazol (not shown) and milrinone (FIG. 8), on cAMP hydrolytic activity in cytosolic and microsomal fractions of human myocardium were examined. These drugs had more potent effects in microsomal fractions (FIG. 8).
The PDE3 inhibitor milrinone was used to quantify PDE3 cAMP- and cGMP-hydrolytic activity in lysates of Sf9 cells expressing recombinant PDE3A isoforms and in cytosolic and salt-washed microsomal fractions of human myocardium (Table 5). Catalytic activity was measured at 0.1 lam cAMP and cGMP. Milrinone-sensitive activity for tissue fractions was calculated by measuring cyclic nucleotide hydrolysis inhibited by milrinone at concentrations equal to its IC50
values for cAMP and cGMP hydrolysis by recombinant PDE3A1, and dividing by 0.5.
|TABLE 5 |
|Milrinone-sensitive cAMP and cGMP hydrolytic activity in subcellular fractions |
|of human myocardium |
| ||milrinone-sensitive ||milrinone-sensitive || |
| ||cAMP hydrolytic ||cGMP hydrolytic ||ratio milrinone-sensitive |
| ||activity, ||activity, ||cAMP/cGMP hydrolytic |
|Preparation ||pmol/mg/min ||pmol/mg/min ||activity |
|rtPDE3A1 (full ORF) ||1754 ||405 ||4.3 |
|cytosolic fraction, human ||169 ||75 ||2.3 |
|salt-washed microsomes, ||64 ||10 ||6.4 |
|human myocardium |
|rtPDE3AΔ5 (aa 623-1141) ||1408 ||346 ||4.1 |
The ratio of milrinone-sensitive cAMP/cGMP hydrolytic activity in cytosolic fractions was lower than that observed with full-length recombinant PDE3A, while the ratio in microsomal fractions was higher. To determine if these differences were the result of N-terminal deletions, the same studies were performed on lysates of Sf9 cells expressing a recombinant PDE3A from which the N-terminus had been deleted (rtPDE3AΔ5). N-terminal truncation did not affect the cAMP/cGMP activity ratio.
The higher ratio of milrinone-sensitive cAMP/cGMP hydrolytic activity in the microsomal fraction relative to that seen in recombinant PDE3 isoforms suggests that localization to intracellular membranes increases the selectivity of PDE3 isoforms for cAMP, possibly resulting from the interaction of membrane-bound PDE3 with other proteins.
The contribution of PDE3 isoforms to compartmental regulation of cyclic nucleotide hydrolysis was examined in subcellular preparations from native human myocardium and cultured pulmonary artery myocytes. The results are presented in the Table 6.
PDE3 comprises the majority of the total cAMP hydrolytic activity in microsomal fractions of human myocardium at both low and high cAMP concentrations. It comprises a smaller but significant fraction of cAMP hydrolytic activity in cytosolic fractions of these cells, probably reflecting the larger presence of other cAMP phosphodiesterases in the cytosol. In cultured pulmonary artery myocytes, these findings are reversed. PDE3 contributes less to membrane-bound cAMP hydrolytic activity but more to cytosolic cAMP hydrolytic activity.
PDE3 comprises a large portion of the total cGMP hydrolytic activity in microsomal fractions of human myocardium at low but not at high cGMP concentrations. This likely reflects the presence of other lower affinity cGMP phosphodiesterases in these fractions.
|TABLE 6 |
|Milrinone-sensitive cAMP and cGMP hydrolytic |
|activities in subcellular |
|fractions of human myocardium and cultured |
|human pulmonary artery myocytes |
|% of total cAMP and cGMP hydrolytic activities |
|in the identified fractions |
| ||0.1 μM ||1.0 μM ||0.1 μM ||1.0 μM |
| ||cAMP ||cAMP ||cGMP ||cGMP |
| || |
| ||Myocardium, ||52% ||54% ||10% ||9% |
| ||cytosol |
| ||Myocardium, ||67% ||73% ||42% ||13% |
| ||microsomes |
| ||Pulmonary artery, ||40% ||31% ||19% ||4% |
| ||cytosol |
| ||Pulmonary artery, ||24% ||39% ||14% ||9% |
| ||microsomes |
| || |
- Example 9
Phosphorylation Sites and Effects of Phosphorylation on PDE3A Isoforms
PDE3 contributes relatively little to cGMP hydrolytic activity in cytosolic fractions of human myocardium. PDE3 comprises a surprisingly small portion of the total cGMP hydrolytic activity in both microsomal and cytosolic fractions of pulmonary artery myocytes at both low and high concentrations of substrate. The fact that PDE3's contribute less to total cGMP hydrolytic activity than to total cAMP hydrolytic activity in subcellular fractions of these cells, taken in the context of the fact that competitive PDE3 inhibitors inhibit cAMP hydrolytic activity more potently than they inhibit cGMP hydrolytic activity of PDE3 (see Table 7), suggest that the clinical effects of currently-available competitive PDE3 inhibitors are likely to be mediated to a greater degree by increases in cAMP content than by increases in cGMP content in both cardiac and vascular myocytes. This conclusion cannot be extrapolated to agents that may inhibit PDE3 activity through non-competitive mechanisms proposed herein. The latter may change the profile of cellular actions of PDE3 inhibition, representing an additional possible benefit to the approaches proposed over currently available therapies.
|TABLE 7 |
|IC50's for inhibition of rtPDE3A by milrinone |
|Substrate concentration ||0.1 μM cAMP ||1.0 μM cAMP ||0.1 μM cGMP ||1.0 μM cGMP |
|IC50 ||0.9 μM ||6.0 μM ||2.4 μM ||23 μM |
The phosphorylation sites on PDE3A-136 were localized by labeling studies to amino acid residues 288-294 (P1 site), 309-312 (P2 site) and 435-438 (P3 site). The P2 and P3 sites on PDE3A-136 only contain one serine residue each and the phosphorylated residue is unambiguous (FIG. 11). The P1 site contains multiple serine residues and it is not certain at present which is phosphorylated.
Differences with respect to the presence of PK-A and PK-B sites in the different isoforms of PDE3 indicate differences in regulation by phosphorylation. PDE3A-136 and PDE3B-137 contain sites P1, P2 and P3 and are thus potentially subject to regulation by both PK-A and PK-B (Table 4). PDE3A-118 contains only P2 and P3 and can thus be regulated only by PK-A (Table 4). PDE3A-94 contains none of these phosphorylation sites therefore its activity can be regulated by neither PK-A nor PK-B. These N-terminal sequence differences may lead to differences in regulation by other interacting partners.
The effects of phosphorylation at the P3 site of PDE3A, along with the apparently equivalent site on PDE3B-137, on phosphodiesterase catalytic activity are shown in Table 8. Flag-tagged rtPDE3B-137 isoforms (full ORF's) were prepared with mutations at P3, one of the two PK-A sites (FIG. 4). These included a constitutively nonphosphorylated form, in which Ser421 was mutated to alanine (“S421A”) and a form that acted as if it were constitutively phosphorylated, in which Ser421 was mutated to aspartic acid (“S421D”). The charged group on the end of the aspartate side chain resembles a phosphate group in its effect on phosphodiesterase activity. These recombinant isoforms were used, together with the corresponding wild-type rtPDE3B, to examine the effects of phosphorylation at site P3 on catalytic activity and inhibitor sensitivity. Catalytic activity of PDE3 was measured in detergent-solubilized lysates of Sf21 cells expressing Flag-tagged rtPDE3 isoforms (full-length ORFs). Values for Vmax and Km were calculated by nonlinear regression (first-order kinetics). Preparations were diluted so that each contained equal concentrations of immunoreactive PDE3 as determined by quantitative Western blotting with anti-Flag antibodies. The three isoforms of rtPDE3B were observed to have comparable catalytic activity toward cAMP and cGMP (Table 8). The three isoforms also exhibited similar sensitivity to inhibition by cilostazol (data not shown). This suggests that phosphorylation at P3 has little if any direct effect on enzyme activity.
|TABLE 8 |
|Effect of ser421 mutations on catalytic activity of PDE3B |
| ||cAMP hydrolysis ||cGMP hydrolysis |
|rtPDE3B ||Vmax ||Km ||Vmax ||Km |
|isoform ||pmol/min/ml ||μM ||pmol/min/ml ||μM |
|wild type ||977 ± 53 ||0.15 ± 0.03 ||325 ± 18 ||0.09 ± 0.02 |
|S421A || 968 ± 144 ||0.21 ± 0.09 ||300 ± 16 ||0.07 ± 0.02 |
|S421D ||809 ± 12 ||0.17 ± 0.01 ||241 ± 42 ||0.07 ± 0.04 |
- Example 10
Site-specific Mutations and Phosphorylation
The rtPDE3B's were used to study the effects of phosphorylation by PK-A at other sites. Phosphorylation of these isoforms with PK-A caused a much greater stimulation of activity in S421D than in S421A or the wild-type rtPDE3B (FIG. 9). These results indicate an interaction between P3 and P2, the upstream PK-A site. Phosphorylation of P3 may increase the stimulation of activity by PK-A by facilitating phosphorylation at P2. The fact that stimulation of the wild-type rtPDE3B has less effect than a Ser→Asp mutation at P3 may reflect incomplete phosphorylation of the latter site. Alternatively, phosphorylation of P3 may potentiate the effect of phosphorylation of P2 on enzyme activity. Another possibility is that phosphorylation of P3 has an inhibitory effect on catalytic activity that is overcome by phosphorylation of P2.
The phosphorylation of PDE3B-137, PDE3A-136 and PDE3A-118 by PK-A and PK-B is examined using recombinant constructs with thrombin cleavage sites followed by hiS6 tags at the C-terminus. Constructs are expressed in Sf9 cells by infection with baculovirus vector. His6-tagged recombinant proteins are purified by Co 2+-affinity chromatography (Clontech resin) and their his6 tags are removed by thrombin cleavage.
rtPDE3's are phosphorylated by PK-A (Sigma) and PK-B Upstate Biotechnology). Varying concentrations of purified rtPDE3's are incubated in the presence of nanomolar concentrations of kinase, saturating concentrations of [γ-32P]ATP and phosphatase inhibitors. Reaction mixtures are subjected to SDS-PAGE, and 32P incorporation is quantified in excised PDE3 bands following established protocols (Movsesian et al., 1984). Values for Km and Vmax for phosphorylation by PK-A and PK-B are calculated by nonlinear regression and standardized using peptide substrates as controls (Kemptide for PK-A, Crosstide for PK-B).
The use of rtPDE3's with Ser→Ala and Ser→Asp mutations at selected phosphorylation sites allows the isolation of individual sites (by rendering others nonphosphorylatable). Interactions between sites may also be examined. For example, to study the effect of phosphorylation at P2 by PK-A on phosphorylation at P1 by PK-B, rtPDE3's are prepared with Ser→Ala and Ser→Asp mutations at P2 and P3. The effects on Km and Vmax for phosphorylation by PK-B at P1 are examined.
Non-physiologic artifacts may be induced using Ser→Asp mutations. For example, they may mimic phosphorylation at a site that is not phosphorylated in vivo in the cell of interest. To address this problem, the phosphorylation of specific sites in aortic myocytes and HL-1 cells transfected with tagged rtPDE3's is examined. To examine phosphorylation at P1, HL-1 cells are transfected with PDE3 constructs with Ser→Ala and Ser→Asp mutations at P2 and P3, using HL-1 cells transfected with Ser→Ala mutations at P1 as a negative control. Cells are preincubated with 32PO4 3− and exposed to β1- and β2-adrenergic receptor agonists, forskolin, PGE2 and IBMX (to activate PK-A) and/or IGF-1±wortmannin (to activate PI3-K, which phosphorylates and activates PK-B). PDE3 is immunoprecipitated from the resulting cellular fractions with anti-Tag antibodies and subjected to SDS-PAGE and autoradiography to determine whether phosphorylation at P1 has occurred and is influenced by phosphorylation at other sites. Quantitative western blotting is then performed to normalize 32p incorporation to immunoreactive PDE3. This approach may be used to determine whether phosphorylation of one site affects phosphorylation of another in vivo (cultured cells). This approach has been validated in adipocytes where the sites phosphorylated in transfected proteins have been determined to be the same as those phosphorylated in native proteins (Kitamura, et al., 1999).
- Example 11
Effects of Phosphorylation on Intracellular Localization
Two similar approaches may be performed to validate phosphorylation in cultured myocytes. First, antibodies are raised to synthetic peptides corresponding to phosphorylated P1, P2 and P3 domains. The studies described above are repeated in nontransfected cells (without radiolabeling). SDS-PAGE is performed on cell homogenates and the phosphospecific antibodies are used to confirm or refute phosphorylation at individual sites by Western blotting. The same studies may be performed after preincubation with 32PO4 3−. Native PDE3's are immunoprecipitated from cellular homogenates with anti-CT antibodies. SDS-PAGE is performed on these native proteins and the PDE bands are excised. The protein is extracted from the gel material and limited proteolysis with trypsin, chymotrypsin, CNBr and/or V8 is performed. The resulting peptide fragments are resolved via two-dimensional mapping, using two-dimensional peptide maps of mutagenized rtPDE3's phosphorylated in vitro as controls. Comparison thereof reveals which sites are phosphorylated in the HL-1 cells.
The role of the N-terminus in intracellular targeting was elucidated through an approach that involved the transfection of cultured cells with rtPDE3 constructs. This approach may be expanded by stably transfecting cultured aortic myocytes (Clonetics) with his6- or Flag-tagged PDE3B-137- and PDE3A-118-derived constructs with Ser→Ala and Ser→Asp mutations at the three phosphorylation sites identified herein. PDE3A-94 is not included because it does not appear to contain any of the phosphorylation sites.
The protocol for stable transfection uses the vector pCDNA 3.1 (Invitrogen). This vector is driven by a CMV promoter, includes a neomycin resistance element for selection and adds a C-terminal myc-his6 tag to the expressed protein. The choice of stable rather than transient transfection is based on the higher levels of recombinant protein expression observed in stable transformants (not shown). The intracellular localization of rtPDE3 isoforms is determined by indirect immunofluorescence using fluorophore-tagged anti-his6 or anti-Flag antibodies. Co-localization relies on the use of antibodies to markers for different intracellular membranes. Phosphorylation does not induce translocation of PDE3B-137, as it contains the transmembrane helices of NHR1 and is therefore likely to be an intrinsic membrane protein. However, some combinations of Ser→Asp mutations induce a translocation of PDE3A-118 from intracellular membranes to the cytosol.
- Example 12
Indirect Immunofluorescence and Intracellular Localization
The results of these studies may not be applicable to cardiac myocytes, since the PDE3 isoforms are not identical and the intracellular targeting mechanisms may differ. For this reason, the studies described above may be repeated in cardiac myocytes or cells derived from cardiac myocytes using PDE3A-136 instead of PDE3B-137.
The effects of phosphorylation of the sites P1, P2 and P3 on the membrane targeting domains NHR1 and NHR2 and intracellular localization were studied. The role of the N-terminus of PDE3 in intracellular targeting was elucidated by transfecting cultured cells with rtPDE3 constructs and visualizing the intracellular localization of these rtPDE3 constructs by indirect immunofluorescence. COS-7 cells were transfected with PDE3A and PDE3B constructs with C-terminal Flag-tags and varying N-terminal deletions, and localization was visualized using fluorescein-labeled anti-Flag antibodies. Constructs containing NHR1 were found to be membrane-bound (not shown). Constructs lacking NHR1 but containing NHR2 were partially membrane-bound and partially cytosolic and constructs lacking both NHR1 and NHR2 were exclusively cytosolic (not shown). This distribution corresponds to the distribution of native PDE3's in human myocardium and aortic myocytes.
To extend this approach, cultured aortic myocytes (Clonetics, East Rutherford, N.J.) may be transfected with Flag-tagged PDE3B-137- and PDE3A-118-derived constructs with Ser Ala and Ser Asp mutations at the P1, P2 and P3 PK-A and PK-B phosphorylation sites. Stable transfection utilizes the transcription vector pCDNA 3.1 (Invitrogen, Carlsbad, Calif.). The pCDNA vector is driven by a CMV promoter, includes a neomycin resistance element for selection, and adds a C-terminal Flag tag to the expressed protein. The intracellular localization of rtPDE3 isoforms with mutagenized phosphorylation sites may be determined by indirect immunofluorescence using fluorophore-tagged anti-Flag antibodies. Co-localization relies on the use of commercially available antibodies to markers for different intracellular membranes.
- Example 13
Results in vascular myocytes may not be applicable to cardiac myocytes. The PDE3 isoforms in the two cell types are not identical, and the intracellular targeting mechanisms may be different. For this reason, the above studies may be repeated in HL-1 cells, an immortalized cell line derived from atrial myocytes (Claycomb, et al., 1998). Western blotting indicates that the representation of PDE3 isoforms in subcellular fractions prepared from these cells is similar to that seen in preparations from human left ventricular myocardium, making these cells particularly suitable for these experiments. Transfections of HL-1 cells is performed with PDE3A-136- rather than PDE3B-derived constructs to reflect the different patterns of cellular expression. This transfection may be transient or stable. A high percentage of transfection efficiency with PDE3 constructs using transient transfection obviates the need for stable transfection of rtPDE3 isoforms.
The interactions of PK-B with PDE3B were examined. Microsomal fractions of 3T3 adipocytes (which express PDE3B) were solubilized with NP-40 and fractionated by gel filtration. Western blotting showed the presence of separate peaks for PDE3B and PK-B, but some of the PK-B was found in the PDE3B peak (not shown). An association between PK-B and PDE3B was confirmed by the ability of anti-PDE3B antibodies to co-immunoprecipitate the two proteins in the PDE3B peak (not shown). Treatment with insulin increased the phosphorylation of PK-B and appeared to increase the percentage of PK-B co-purifying with PDE3B (not shown). These results suggest that PK-B and PDE3B form stable complexes in vivo, either by direct interaction or by co-interaction with another protein.
Detergent-solubilized lysates of Sf9 cells expressing rtPK-B were mixed with detergent-solubilized lysates of Sf9 cells expressing one of two Flag-tagged forms of PDE3B. The first isoform of PDE3B contained its full ORF. The second lacked the N-terminal 604 amino acids containing the NHR1, NHR2 and the three phosphorylation sites. PK-B could be co-immunoprecipitated with anti-Flag antibodies in the presence of the fill-length rtPDE3B but not in the presence of the N-terminal-deleted form (FIG. 10), confirming the role of the N-terminus of PDE3B in its association with PK-B.
- Example 14
Identification of PDE3 Kinases, Phosphatases and Binding Peptides/Interacting Partners
The addition of Flag-tagged rtPDE3B to 3T3 lysates allowed the co-immunoprecipitation of AKAP220, which co-localizes PK-A and PP1 (Schillace et al., 2001). This indicates that interactions with other proteins serves to localize PDE3 to specific signaling modules, and suggests that blocking these interactions will alter the function of PDE3.
- Preparation of Phage-displayed cDNA Libraries
Purified rtPDE3's may be used as affinity ligands to identify PDE3-binding proteins (“PDE3-BP's”) by interaction cloning from phage-displayed myocardial and vascular smooth muscle cDNA libraries. This approach involves two basic steps: preparation of phage-displayed cDNA libraries and biopanning with rtPDE3.
cDNA inserts from commercially available human cardiac (Xba I-(dT)15-primed) and aortic (oligo(dT) and random-primed) libraries (Clontech, Palo Alto, Calif.) are PCR-amplified using vector-derived primers (λTrip1Ex for cardiac, λgt10 for aortic) with unique restriction sites. These libraries have been used to clone PDE3 isoforms, which are expressed in relatively low abundance. PCR products are size-fractionated on agarose gels. Products greater than 500 nucleotides in length are purified by agarose gel electrophoresis and ligated into the genes of phage coat proteins using unique restriction sites. Proteins or protein fragments encoded by the cDNA inserts are displayed on the phage surface.
- Biopanning with rtPDE3
Two phage with different reproductive biologies are used. One is M13, a non-lytic phage that is secreted after assembly in the bacterial periplasm. cDNA inserts up to 1000 amino acids in length can be expressed as C-terminal fusions to the pVI coat protein of M13. The protocols used are as disclosed in Fransen et al. (1999). The same vectors and protocols are used to insert human cardiac and aortic cDNA libraries into pVI. The second phage is T7 (Novagen, Madison, Wis.). This phage, being lytic, is processed quite differently from M13, so that cDNA inserts that may interfere with M13 function are not likely to affect T7 (and vice versa). T7 is capable of displaying cDNA products up to 1200 amino acids in length. Methods for its use have been disclosed in Zozulya et al. (1999).
Phage with cDNA inserts are incubated with rtPDE3's that are immobilized either directly onto polystyrene wells or indirectly by binding of C-terminal hiS6 tags to anti-his6 mAb, followed by immunoprecipitation. Phage whose cDNA inserts encode full-length or truncated PDE3-BP's are co-immobilized with PDE3, then eluted and amplified in E. coli. Each round of this procedure yields a phage library enriched in cDNAs encoding PDE3-binding proteins. Biopanning is repeated through several iterations until the titer of phage binding to immobilized PDE3 is ten-fold above background (phage binding to wells in the absence of PDE3), at which point individual phage colonies are cloned and their cDNA inserts sequenced.
Phage are biopanned with rtPDE3s. rtPDE3A-118 and rtPDE3A-94 are used for both cardiac and aortic libraries. PDE3B-137 and PDE3A-136 are used exclusively for aortic and cardiac libraries, respectively. Thiophosphorylated rtPDE3 are prepared with PK-A and/or PK-B and ATPγS for use as bait in parallel experiments to select proteins that bind preferentially to phosphorylated PDE3s, for example, phosphatases. Phosphothioesters are resistant to dephosphorylation and thiophosphorylated proteins therefore bind stably to protein phosphatases.
Cloned cDNA sequences identified by biopanning may be used to search protein databases and identify full-length binding proteins for PDE3.
- Example 15
Characterization of Binding Interactions and Effects on PDE3 Function
Confirmation of Binding of Cloned Prospective PDE3-BP 's to PDE3
The skilled artisan will realize that the methods discussed above could be used to identify novel isoform selective inhibitors or activators of PDE3. Purified isoform proteins are used as ligands for biopanning general phage display libraries comprising random nucleic acid sequences encoding short peptides. Phage that bind with relatively high affinity to one or more PDE3 isoforms are selected and their DNA inserts are sequenced. The encoded peptides are chemically synthesized and their ability to activate or inhibit PDE3 catalytic activity or to block or mimic the effect of phosphorylation at P1, P2 or P3 on catalytic activity is examined using standard enzyme analysis. The effect of identified activators or inhibitors on each PDE3 isoform is determined and isoform selective compounds are identified. Use of site specific mutagenized isoforms that are designed to be constitutively unphosphorylatable or to mimic constitutively phosphorylated residues at P1, P2 and P3 identifies activators or inhibitors that are selective for phosphorylated or dephosphorylated variants of each isoform.
- Characterization of Binding Interactions and Effects on PDE3 Function
Binding interactions are confirmed by co-immunoprecipitation, which can occur in any of four ways. First, native PDE is immunoprecipitated from lysates of cardiac and aortic myocytes using anti-PDE antibodies and co-immunoprecipitation is confirmed via Western blotting using antibodies raised to the cloned PDE-BP. The second method reverses the order of the antibodies used. Thus, antibodies to the cloned PDE3-BP are used for immunoprecipitation and co-immunoprecipitation is confirmed via Western blotting using anti-PDE3 antibodies. Third, aortic myocytes or HL-1 cells are transfected with Flag-tagged rtPDE3-BP's, followed by co-immunoprecipitation and Western blotting with anti-Flag antibodies. Lastly, tagged rtPDE3's and rtPDE3-BP's are expressed by in vitro transcription/translation in reticulocyte lysates or in a baculovirus/Sf9 system. the recombinant proteins are co-incubated and co-immunoprecipitation is tested for as for AKAP-220, a method described elsewhere in this document.
The affinity (KD) of the interaction between PDE3 and various binding proteins or peptides may be determined by ELISA, using immobilized rtPDE3 and rtPDE3-BP's (obtained by expression in E. coli or Sf9/Sf21 cells). The effects of rtPDE3-BP's on the catalytic activity and inhibitor sensitivity of rtPDE3's is determined as described above. The effects of PDE3-BP's on the phosphorylation of rtPDE3's by PK-A and PK-B in vitro is determined as described above. rtPDE3's with Ser→Ala and Ser→Asp mutations are used to determine how phosphorylation at specific sites affects interactions with PDE3-BP's.
Interacting domains of PDE3's and their binding partners are identified by deletional and site-directed mutagenesis of PDE3 and/or PDE3-BPs. Peptides derived from interacting domains are examined for inhibition of PDE3/PDE3-BP interactions. Inhibition of PDE3/PDE3-BP interactions is examined by ELISA or by measuring inhibition of functional correlates of binding. For example, if binding to a PDE3-BP increases the Km of PDE3 for cAMP, the ability of peptides to prevent this increase is determined. Alternatively, peptides that mimic the effects of PDE3-BP's may be PDE3 activators. Peptides in either category are of interest as potential therapeutic agents and may serve as templates for peptidomimetic drugs or reporters for high-throughput screening.
Peptides derived from the phage display experiments derived above are also tested for their ability to either block the binding of PDE3 to PDE3-BP's or to mimic the effect of PDE3-BP's on catalytic activity or inhibitor sensitivity of PDE3.
- Effects of Phosphorylation on Interactions Between PDE3 and PDE3-BP
To quantify the affinity of PDE3 to PDE3-BP, surface plasmon resonance (Biacore, Piscataway, N.J.) using purified rtPDE3's and rtPDE3-BP's (obtained by expression in E. coli or Sf9/Sf21 cells) is performed. Generally, surface plasmon resonance (SPR) uses light reflected from a conducting film at the interface between two media of different refractive index. In this instance, the media are the biological sample and the glass of a sensor chip. The conducting film is a thin layer of gold on the sensor chip surface. When the molecules in the biological sample bind to the surface of the sensor chip, the concentration (and therefore the refractive index) at the chip surface changes and an SPR response is detected. Here, his-tagged rtPDE3's are captured by anti-his monoclonal antibodies immobilized on flow-cell surfaces of biosensor chips. A series of concentrations of rtPDE3-BP's (expressed in Sf9 cells and purified as described above) are superfused thereon and surface plasmon resonance responses are used to determine values for KD.
To determine the effects of phosphorylation at specific sites on interactions between PDE3 and PDE3-BP, surface plasmon resonance experiments are performed as above using rtPDE3's with Ser Ala and Ser Asp mutations at the three phosphorylation sites P1, P2 and P3. The effects of these mutations on the KD of the reaction described above are determined. The kinetics of phosphorylation at P1, P2 and P3 by PK-A and PK-B in the presence and absence of PDE3-BP's are also determined.
The ability of any new PDE3 kinase to phosphorylate P1, P2 and P3 may be examined for PK-A and PK-B, as described above.
The ability of PDE3 phosphatases to dephosphorylate P1, P2 and P3 may also be determined. This entails the use of rtPDE3's with Ser Ala mutations at all but one of the phosphorylation sites. These rtPDE3's are phosphorylated in the presence of [γ-32P]ATP and the appropriate kinase (e.g. PK-A or PK-B). 32p release in the presence of phosphatase is characterized in terms of Vmax and Km. rtPDE3's with Ser Asp mutations are then used to determine the effect of phosphorylation at one site on dephosphorylation at another.
- Identification of the Interacting Domains of PDE3 and PDE3-BP
The effect of PDE3-BP's interactions on catalytic activity, substrate preference, and inhibitor sensitivity is determined by measuring cyclic nucleotide hydrolysis in the absence and presence of PDE3-BP's. Functional K values for PDE3/PDE3-BP's interactions are determined and compared to the KD values determined by surface plasmon resonance.
- Example 16
siRNA Inhibition of PDE3A1
Identification of the interacting domains of PDE3's and PDE3-BP's is done via deletional and site-directed mutagenesis of PDE3 and/or PDE3-BP. Several lines of evidence suggest that compartmentally nonselective increases in intracellular cAMP content in cardiac myocytes have both beneficial and harmful effects in dilated cardiomyopathy. Agents capable of selectively activating or inhibiting individual PDE3 isoforms localized to different intracellular compartments or of selectively affecting activity toward cAMP or cGMP may offer major advantages in therapeutic applications. Peptides that block or interfere with the interaction of PDE3 with PDE3-BP may be used to identify functional consequences in vivo. Alternatively, peptides that mimic the effects of PDE3-BP's may be PDE3 activators. Either category of peptides would be useful tools for studying the function of PDE3 isoforms in vivo and may be of interest as prototypical therapeutic agents. They may serve as templates for peptidomimetic drugs or may be tagged for use as reporters for high throughput screening.
21-nucleotide siRNAs are chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite chemistries (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel-purifed. The siRNA sequence targeting the PDE3A1 mRNA corresponds to the nucleotide sequences −268 to −241 of the human myocardial PDE3A1 cDNA sequence (SEQ ID NO:18; GenBank Accession No. NM000921). That sequence is located in the 5′ untranslated region of the PDE3A1 mRNA (SEQ ID NO:18), and is not present in PDE3A2 (SEQ ID NO:15). It should therefore be specific for inhibition of expression of the PDE3A-136 protein.
- Example 17
Isoform Specific Probe and Antisense Construct
Sf21 cells expressing rtPDE3A1 are grown at 37° C. in grown in TNM-FH media (BD-Pharmingen, San Diego, Calif.). Transfection with 1.0 nM siRNA is performed with Oligofectamine (Life Technologies) as described by the manufacturer. Cells are incubated 20 h after transfection and expression of rtPDE3A1 is assayed by Northern blotting. Transfection with siRNA is observed to result in a complete inhibition of rtPDE3A1 expression in Sf21 cells. Control cells transfected with a random 21 bp siRNA sequence show no effect on rtPDE3A1 expression.
In certain embodiments of the invention, isoform specific probes may be constructed and used, for example to determine the levels of expression of the PDE3 isoforms in different cells or tissues or in response to various putative inhibitors or activators, such as in a high throughput screening assay directed towards mRNAs. Because the downstream (3′) portions of the PDE3A mRNAs (SEQ ID NO:14, SEQ ID NO:15) are apparently identical, the only region available for isoform specific probes and/or antisense constructs are at the 5′ end of the PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18). An exemplary probe specific for the A mRNA encoding the PDE3A-136 isoform protein is disclosed below.
|(SEQ ID NO:13) || |
|TGATCGTTTCTGCCCGTGCTTGTTTTCAACTTGAGCGTGCTAGCCTTTAA || |
The probe sequence corresponds to nucleotides −268 to 189 of PDE3A1 (SEQ ID NO:14, SEQ ID NO:18), where nucleotide 1 starts with the first ATG codon in the largest open reading frame (ORF) of the PDE3A1 cDNA sequence (SEQ ID NO:14). The probe sequence (SEQ ID NO:13) is located primarily in exon 1 of the PDE3A1 mRNA, starting in the 5′ UTR and ending just before the NHR1 sequence. Primers may be used to generate the probe from the PDE3A1 cDNA or to amplify the target sequence from sample RNA, as disclosed below.
| || |
| ||Sense Strand: || || |
| ||TGATCGTTTCTGCCCGTGCTTGTTTTC ||(SEQ ID NO:16) |
| || |
| ||Anti-sense: |
| ||CAGGGAAAGTTTCCGAGAGCTCCGGAG ||(SEQ ID NO:17) |
The skilled artisan will realize that there are many potential uses for the isoform specific probe and primers disclosed above. For example, expression of PDE3A1 could be measured in various cells or tissues in either normal individuals or individuals with a disease state, such as cardiomyopathy and/or pulmonary hypertension. The effects of various putative activators or inhibitors on PDE3A1 expression in intact cells could be determined as part of a high throughput screening assay. Alternatively, an antisense construct, ribozyme and/or siRNA inhibitor could be designed to bind only to PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18). Such an inhibitor would decrease activity of PDE3A-136, while leaving PDE3A-118 and PDE3A-94 activity unaffected. Since SEQ ID NO:13 shows the sequence of part of the PDE3A1 cDNA, the skilled artisan will realize that an antisense constructs would be designed to be complementary, preferably exactly complementary, to part or all of the sequence of SEQ ID NO:13. Such a construct could be designed as a double-stranded DNA sequence that is functionally coupled to a promoter and inserted into an expression vector that can be transfected into a target cell. Expression vectors of use in mammalian cells are well known in the art, as summarized above.
All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the COMPOSITIONS, METHODS and APPARATUS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defmed by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Met Gly Leu Tyr Leu Leu Arg Ala Gly Val Arg Leu Pro Leu Ala Val
1 5 10 15
Ala Leu Leu Ala Ala Cys Cys Gly Gly Glu Ala Leu Val Gln Ile Gly
20 25 30
Leu Gly Val Gly Glu Asp His Leu Leu Ser Leu Pro Ala Ala Gly Val
35 40 45
Val Leu Ser Cys Leu Ala Ala Ala Thr Trp Leu Val Leu Arg Leu Arg
50 55 60
Leu Gly Val Leu Met Ile Ala Leu Thr Ser Ala Val Arg Thr Val Ser
65 70 75 80
Leu Ile Ser Leu Glu Arg Phe Lys Val Ala Trp Arg Pro Tyr Leu Ala
85 90 95
Tyr Leu Ala Gly Val Leu Gly Ile Leu Leu Ala Arg Tyr Val Glu Gln
100 105 110
Ile Leu Pro Gln Ser Ala Glu Ala Ala Pro Arg Glu His Leu Gly Ser
115 120 125
Gln Leu Ile Ala Gly Thr Lys Glu Asp Ile Pro Val Phe Lys Arg Arg
130 135 140
Arg Arg Ser Ser Ser Val Val Ser Ala Glu Met Ser Gly Cys Ser Ser
145 150 155 160
Lys Ser His Arg Arg Thr Ser Leu Pro Cys Ile Pro Arg Glu Gln Leu
165 170 175
Met Gly His Ser Glu Trp Asp His Lys Arg Gly Pro Arg Gly Ser Gln
180 185 190
Ser Ser Gly Thr Ser Ile Thr Val Asp Ile Ala Val Met Gly Glu Ala
195 200 205
His Gly Leu Ile Thr Asp Leu Leu Ala Asp Pro Ser Leu Pro Pro Asn
210 215 220
Val Cys Thr Ser Leu Arg Ala Val Ser Asn Leu Leu Ser Thr Gln Leu
225 230 235 240
Thr Phe Gln Ala Ile His Lys Pro Arg Val Asn Pro Val Thr Ser Leu
245 250 255
Ser Glu Asn Tyr Thr Cys Ser Asp Ser Glu Glu Ser Ser Glu Lys Asp
260 265 270
Lys Leu Ala Ile Pro Lys Arg Leu Arg Arg Ser Leu Pro Pro Gly Leu
275 280 285
Leu Arg Arg Val Ser Ser Thr Trp Thr Thr Thr Thr Ser Ala Thr Gly
290 295 300
Leu Pro Thr Leu Glu Pro Ala Pro Val Arg Arg Asp Arg Ser Thr Ser
305 310 315 320
Ile Lys Leu Gln Glu Ala Pro Ser Ser Ser Pro Asp Ser Trp Asn Asn
325 330 335
Pro Val Met Met Thr Leu Thr Lys Ser Arg Ser Phe Thr Ser Ser Tyr
340 345 350
Ala Ile Ser Ala Ala Asn His Val Lys Ala Lys Lys Gln Ser Arg Pro
355 360 365
Gly Ala Leu Ala Lys Ile Ser Pro Leu Ser Ser Pro Cys Ser Ser Pro
370 375 380
Leu Gln Gly Thr Pro Ala Ser Ser Leu Val Ser Lys Ile Ser Ala Val
385 390 395 400
Gln Phe Pro Glu Ser Ala Asp Thr Thr Ala Lys Gln Ser Leu Gly Ser
405 410 415
His Arg Ala Leu Thr Tyr Thr Gln Ser Ala Pro Asp Leu Ser Pro Gln
420 425 430
Ile Leu Thr Pro Pro Val Ile Cys Ser Ser Cys Gly Arg Pro Tyr Ser
435 440 445
Gln Gly Asn Pro Ala Asp Glu Pro Leu Glu Arg Ser Gly Val Ala Thr
450 455 460
Arg Thr Pro Ser Arg Thr Asp Asp Thr Ala Gln Val Thr Ser Asp Tyr
465 470 475 480
Glu Thr Asn Asn Asn Ser Asp Ser Ser Asp Ile Val Gln Asn Glu Asp
485 490 495
Glu Thr Glu Cys Leu Arg Glu Pro Leu Arg Lys Ala Ser Ala Cys Ser
500 505 510
Thr Tyr Ala Pro Glu Thr Met Met Phe Leu Asp Lys Pro Ile Leu Ala
515 520 525
Pro Glu Pro Leu Val Met Asp Asn Leu Asp Ser Ile Met Glu Gln Leu
530 535 540
Asn Thr Trp Asn Phe Pro Ile Phe Asp Leu Val Glu Asn Ile Gly Arg
545 550 555 560
Lys Cys Gly Arg Ile Leu Ser Gln Val Ser Tyr Arg Leu Phe Glu Asp
565 570 575
Met Gly Leu Phe Glu Ala Phe Lys Ile Pro Ile Arg Glu Phe Met Asn
580 585 590
Tyr Phe His Ala Leu Glu Ile Gly Tyr Arg Asp Ile Pro Tyr His Asn
595 600 605
Arg Ile His Ala Thr Asp Val Leu His Ala Val Trp Tyr Leu Thr Thr
610 615 620
Gln Pro Ile Pro Gly Leu Ser Thr Val Ile Asn Asp His Gly Ser Thr
625 630 635 640
Ser Asp Ser Asp Ser Asp Ser Gly Phe Thr His Gly His Met Gly Tyr
645 650 655
Val Phe Ser Lys Thr Tyr Asn Val Thr Asp Asp Lys Tyr Gly Cys Leu
660 665 670
Ser Gly Asn Ile Pro Ala Leu Glu Leu Met Ala Leu Tyr Val Ala Ala
675 680 685
Ala Met His Asp Tyr Asp His Pro Gly Arg Thr Asn Ala Phe Leu Val
690 695 700
Ala Thr Ser Ala Pro Gln Ala Val Leu Tyr Asn Asp Arg Ser Val Leu
705 710 715 720
Glu Asn His His Ala Ala Ala Ala Trp Asn Leu Phe Met Ser Arg Pro
725 730 735
Glu Tyr Asn Phe Leu Ile Asn Leu Asp His Val Glu Phe Lys His Phe
740 745 750
Arg Phe Leu Val Ile Glu Ala Ile Leu Ala Thr Asp Leu Lys Lys His
755 760 765
Phe Asp Phe Val Ala Lys Phe Asn Gly Lys Val Asn Asp Asp Val Gly
770 775 780
Ile Asp Trp Thr Asn Glu Asn Asp Arg Leu Leu Val Cys Gln Met Cys
785 790 795 800
Ile Lys Leu Ala Asp Ile Asn Gly Pro Ala Lys Tyr Lys Glu Leu His
805 810 815
Leu Gln Trp Thr Asp Gly Ile Val Asn Glu Phe Tyr Glu Gln Gly Asp
820 825 830
Glu Glu Ala Ser Leu Gly Leu Pro Ile Ser Pro Phe Met Asp Arg Ser
835 840 845
Ala Pro Gln Leu Ala Asn Leu Gln Glu Ser Phe Ile Ser His Ile Val
850 855 860
Gly Pro Leu Cys Asn Ser Tyr Asp Ser Ala Gly Leu Met Pro Gly Lys
865 870 875 880
Trp Val Glu Asp Ser Asp Glu Ser Gly Asp Thr Asp Asp Pro Glu Glu
885 890 895
Glu Glu Glu Glu Ala Pro Ala Pro Asn Glu Glu Glu Thr Cys Glu Asn
900 905 910
Asn Glu Ser Pro Lys Lys Lys Thr Phe Lys Arg Arg Lys Ile Tyr Cys
915 920 925
Gln Ile Thr Gln His Leu Leu Gln Asn His Lys Met Trp Lys Lys Val
930 935 940
Ile Glu Glu Glu Gln Arg Leu Ala Gly Ile Glu Asn Gln Ser Leu Asp
945 950 955 960
Gln Thr Pro Gln Ser His Ser Ser Glu Gln Ile Gln Ala Ile Lys Glu
965 970 975
Glu Glu Glu Glu Lys Gly Lys Pro Arg Gly Glu Glu Ile Pro Thr Gln
980 985 990
Lys Pro Asp Gln
Met Ser Gly Cys Ser Ser Lys Ser His Arg Arg Thr Ser Leu Pro Cys
1 5 10 15
Ile Pro Arg Glu Gln Leu Met Gly His Ser Glu Trp Asp His Lys Arg
20 25 30
Gly Pro Arg Gly Ser Gln Ser Ser Gly Thr Ser Ile Thr Val Asp Ile
35 40 45
Ala Val Met Gly Glu Ala His Gly Leu Ile Thr Asp Leu Leu Ala Asp
50 55 60
Pro Ser Leu Pro Pro Asn Val Cys Thr Ser Leu Arg Ala Val Ser Asn
65 70 75 80
Leu Leu Ser Thr Gln Leu Thr Phe Gln Ala Ile His Lys Pro Arg Val
85 90 95
Asn Pro Val Thr Ser Leu Ser Glu Asn Tyr Thr Cys Ser Asp Ser Glu
100 105 110
Glu Ser Ser Glu Lys Asp Lys Leu Ala Ile Pro Lys Arg Leu Arg Arg
115 120 125
Ser Leu Pro Pro Gly Leu Leu Arg Arg Val Ser Ser Thr Trp Thr Thr
130 135 140
Thr Thr Ser Ala Thr Gly Leu Pro Thr Leu Glu Pro Ala Pro Val Arg
145 150 155 160
Arg Asp Arg Ser Thr Ser Ile Lys Leu Gln Glu Ala Pro Ser Ser Ser
165 170 175
Pro Asp Ser Trp Asn Asn Pro Val Met Met Thr Leu Thr Lys Ser Arg
180 185 190
Ser Phe Thr Ser Ser Tyr Ala Ile Ser Ala Ala Asn His Val Lys Ala
195 200 205
Lys Lys Gln Ser Arg Pro Gly Ala Leu Ala Lys Ile Ser Pro Leu Ser
210 215 220
Ser Pro Cys Ser Ser Pro Leu Gln Gly Thr Pro Ala Ser Ser Leu Val
225 230 235 240
Ser Lys Ile Ser Ala Val Gln Phe Pro Glu Ser Ala Asp Thr Thr Ala
245 250 255
Lys Gln Ser Leu Gly Ser His Arg Ala Leu Thr Tyr Thr Gln Ser Ala
260 265 270
Pro Asp Leu Ser Pro Gln Ile Leu Thr Pro Pro Val Ile Cys Ser Ser
275 280 285
Cys Gly Arg Pro Tyr Ser Gln Gly Asn Pro Ala Asp Glu Pro Leu Glu
290 295 300
Arg Ser Gly Val Ala Thr Arg Thr Pro Ser Arg Thr Asp Asp Thr Ala
305 310 315 320
Gln Val Thr Ser Asp Tyr Glu Thr Asn Asn Asn Ser Asp Ser Ser Asp
325 330 335
Ile Val Gln Asn Glu Asp Glu Thr Glu Cys Leu Arg Glu Pro Leu Arg
340 345 350
Lys Ala Ser Ala Cys Ser Thr Tyr Ala Pro Glu Thr Met Met Phe Leu
355 360 365
Asp Lys Pro Ile Leu Ala Pro Glu Pro Leu Val Met Asp Asn Leu Asp
370 375 380
Ser Ile Met Glu Gln Leu Asn Thr Trp Asn Phe Pro Ile Phe Asp Leu
385 390 395 400
Val Glu Asn Ile Gly Arg Lys Cys Gly Arg Ile Leu Ser Gln Val Ser
405 410 415
Tyr Arg Leu Phe Glu Asp Met Gly Leu Phe Glu Ala Phe Lys Ile Pro
420 425 430
Ile Arg Glu Phe Met Asn Tyr Phe His Ala Leu Glu Ile Gly Tyr Arg
435 440 445
Asp Ile Pro Tyr His Asn Arg Ile His Ala Thr Asp Val Leu His Ala
450 455 460
Val Trp Tyr Leu Thr Thr Gln Pro Ile Pro Gly Leu Ser Thr Val Ile
465 470 475 480
Asn Asp His Gly Ser Thr Ser Asp Ser Asp Ser Asp Ser Gly Phe Thr
485 490 495
His Gly His Met Gly Tyr Val Phe Ser Lys Thr Tyr Asn Val Thr Asp
500 505 510
Asp Lys Tyr Gly Cys Leu Ser Gly Asn Ile Pro Ala Leu Glu Leu Met
515 520 525
Ala Leu Tyr Val Ala Ala Ala Met His Asp Tyr Asp His Pro Gly Arg
530 535 540
Thr Asn Ala Phe Leu Val Ala Thr Ser Ala Pro Gln Ala Val Leu Tyr
545 550 555 560
Asn Asp Arg Ser Val Leu Glu Asn His His Ala Ala Ala Ala Trp Asn
565 570 575
Leu Phe Met Ser Arg Pro Glu Tyr Asn Phe Leu Ile Asn Leu Asp His
580 585 590
Val Glu Phe Lys His Phe Arg Phe Leu Val Ile Glu Ala Ile Leu Ala
595 600 605
Thr Asp Leu Lys Lys His Phe Asp Phe Val Ala Lys Phe Asn Gly Lys
610 615 620
Val Asn Asp Asp Val Gly Ile Asp Trp Thr Asn Glu Asn Asp Arg Leu
625 630 635 640
Leu Val Cys Gln Met Cys Ile Lys Leu Ala Asp Ile Asn Gly Pro Ala
645 650 655
Lys Tyr Lys Glu Leu His Leu Gln Trp Thr Asp Gly Ile Val Asn Glu
660 665 670
Phe Tyr Glu Gln Gly Asp Glu Glu Ala Ser Leu Gly Leu Pro Ile Ser
675 680 685
Pro Phe Met Asp Arg Ser Ala Pro Gln Leu Ala Asn Leu Gln Glu Ser
690 695 700
Phe Ile Ser His Ile Val Gly Pro Leu Cys Asn Ser Tyr Asp Ser Ala
705 710 715 720
Gly Leu Met Pro Gly Lys Trp Val Glu Asp Ser Asp Glu Ser Gly Asp
725 730 735
Thr Asp Asp Pro Glu Glu Glu Glu Glu Glu Ala Pro Ala Pro Asn Glu
740 745 750
Glu Glu Thr Cys Glu Asn Asn Glu Ser Pro Lys Lys Lys Thr Phe Lys
755 760 765
Arg Arg Lys Ile Tyr Cys Gln Ile Thr Gln His Leu Leu Gln Asn His
770 775 780
Lys Met Trp Lys Lys Val Ile Glu Glu Glu Gln Arg Leu Ala Gly Ile
785 790 795 800
Glu Asn Gln Ser Leu Asp Gln Thr Pro Gln Ser His Ser Ser Glu Gln
805 810 815
Ile Gln Ala Ile Lys Glu Glu Glu Glu Glu Lys Gly Lys Pro Arg Gly
820 825 830
Glu Glu Ile Pro Thr Gln Lys Pro Asp Gln
Met Met Thr Leu Thr Lys Ser Arg Ser Phe Thr Ser Ser Tyr Ala Ile
1 5 10 15
Ser Ala Ala Asn His Val Lys Ala Lys Lys Gln Ser Arg Pro Gly Ala
20 25 30
Leu Ala Lys Ile Ser Pro Leu Ser Ser Pro Cys Ser Ser Pro Leu Gln
35 40 45
Gly Thr Pro Ala Ser Ser Leu Val Ser Lys Ile Ser Ala Val Gln Phe
50 55 60
Pro Glu Ser Ala Asp Thr Thr Ala Lys Gln Ser Leu Gly Ser His Arg
65 70 75 80
Ala Leu Thr Tyr Thr Gln Ser Ala Pro Asp Leu Ser Pro Gln Ile Leu
85 90 95
Thr Pro Pro Val Ile Cys Ser Ser Cys Gly Arg Pro Tyr Ser Gln Gly
100 105 110
Asn Pro Ala Asp Glu Pro Leu Glu Arg Ser Gly Val Ala Thr Arg Thr
115 120 125
Pro Ser Arg Thr Asp Asp Thr Ala Gln Val Thr Ser Asp Tyr Glu Thr
130 135 140
Asn Asn Asn Ser Asp Ser Ser Asp Ile Val Gln Asn Glu Asp Glu Thr
145 150 155 160
Glu Cys Leu Arg Glu Pro Leu Arg Lys Ala Ser Ala Cys Ser Thr Tyr
165 170 175
Ala Pro Glu Thr Met Met Phe Leu Asp Lys Pro Ile Leu Ala Pro Glu
180 185 190
Pro Leu Val Met Asp Asn Leu Asp Ser Ile Met Glu Gln Leu Asn Thr
195 200 205
Trp Asn Phe Pro Ile Phe Asp Leu Val Glu Asn Ile Gly Arg Lys Cys
210 215 220
Gly Arg Ile Leu Ser Gln Val Ser Tyr Arg Leu Phe Glu Asp Met Gly
225 230 235 240
Leu Phe Glu Ala Phe Lys Ile Pro Ile Arg Glu Phe Met Asn Tyr Phe
245 250 255
His Ala Leu Glu Ile Gly Tyr Arg Asp Ile Pro Tyr His Asn Arg Ile
260 265 270
His Ala Thr Asp Val Leu His Ala Val Trp Tyr Leu Thr Thr Gln Pro
275 280 285
Ile Pro Gly Leu Ser Thr Val Ile Asn Asp His Gly Ser Thr Ser Asp
290 295 300
Ser Asp Ser Asp Ser Gly Phe Thr His Gly His Met Gly Tyr Val Phe
305 310 315 320
Ser Lys Thr Tyr Asn Val Thr Asp Asp Lys Tyr Gly Cys Leu Ser Gly
325 330 335
Asn Ile Pro Ala Leu Glu Leu Met Ala Leu Tyr Val Ala Ala Ala Met
340 345 350
His Asp Tyr Asp His Pro Gly Arg Thr Asn Ala Phe Leu Val Ala Thr
355 360 365
Ser Ala Pro Gln Ala Val Leu Tyr Asn Asp Arg Ser Val Leu Glu Asn
370 375 380
His His Ala Ala Ala Ala Trp Asn Leu Phe Met Ser Arg Pro Glu Tyr
385 390 395 400
Asn Phe Leu Ile Asn Leu Asp His Val Glu Phe Lys His Phe Arg Phe
405 410 415
Leu Val Ile Glu Ala Ile Leu Ala Thr Asp Leu Lys Lys His Phe Asp
420 425 430
Phe Val Ala Lys Phe Asn Gly Lys Val Asn Asp Asp Val Gly Ile Asp
435 440 445
Trp Thr Asn Glu Asn Asp Arg Leu Leu Val Cys Gln Met Cys Ile Lys
450 455 460
Leu Ala Asp Ile Asn Gly Pro Ala Lys Tyr Lys Glu Leu His Leu Gln
465 470 475 480
Trp Thr Asp Gly Ile Val Asn Glu Phe Tyr Glu Gln Gly Asp Glu Glu
485 490 495
Ala Ser Leu Gly Leu Pro Ile Ser Pro Phe Met Asp Arg Ser Ala Pro
500 505 510
Gln Leu Ala Asn Leu Gln Glu Ser Phe Ile Ser His Ile Val Gly Pro
515 520 525
Leu Cys Asn Ser Tyr Asp Ser Ala Gly Leu Met Pro Gly Lys Trp Val
530 535 540
Glu Asp Ser Asp Glu Ser Gly Asp Thr Asp Asp Pro Glu Glu Glu Glu
545 550 555 560
Glu Glu Ala Pro Ala Pro Asn Glu Glu Glu Thr Cys Glu Asn Asn Glu
565 570 575
Ser Pro Lys Lys Lys Thr Phe Lys Arg Arg Lys Ile Tyr Cys Gln Ile
580 585 590
Thr Gln His Leu Leu Gln Asn His Lys Met Trp Lys Lys Val Ile Glu
595 600 605
Glu Glu Gln Arg Leu Ala Gly Ile Glu Asn Gln Ser Leu Asp Gln Thr
610 615 620
Pro Gln Ser His Ser Ser Glu Gln Ile Gln Ala Ile Lys Glu Glu Glu
625 630 635 640
Glu Glu Lys Gly Lys Pro Arg Gly Glu Glu Ile Pro Thr Gln Lys Pro
645 650 655
cttcatctct cacattgtgg ggcctctgtg 30
tttgcggccg cctcgagtta tttatcatca tcatctttat aatcctggtc tggcttttgg 60
ggaataatcc agtgctgctg accctcacca aaagcagatc c 41
taatacgact cactataggg agtgaagagg gcaccctata ccatggcag 49
taatacgact cactataggg ttcagtctcc tgtgtgcctt cttctggatg 50
taatacgact cactataggg gaagcgctcg tccagattgg gctgggc 47
taatacgact cactataggg tggagacctt acctggcgta cctggcc 47
taatacgact cactataggg actgcaggaa gcaccttcat ccagtcc 47
tttttttttt tttttttttt tcactggtct ggcttttggg ttggtat 47
tgatcgtttc tgcccgtgct tgttttcaac ttgagcgtgc tagcctttaa cttgaagaag 60
tctcattgga gcatctagca ttctccagga gttattcgaa agctgaaact ttcagtggat 120
tgtgggcctg gggagaagaa ggattccgag ggtggaattg ggaagagcgt gcgtgcgtgt 180
gtgtgtgtgt gtgtgtgcgc gcgcgcgtgg gtcggggcgg gggcgtcggg gggccactgg 240
gaattcagtg aagagggcac cctataccat ggcagtgccc ggcgacgctg cacgagtcag 300
gaacaagccc gtccacagtg gggtgagtca agcccccacg gcgggccggg actgccacca 360
tcgtgcggac cccgcatcgc cgcgggactc gggctgccgt ggctgctggg gagacctggt 420
gctgcagccg ctccggagct ctcggaaact ttccctg 457
atggcagtgc ccggcgacgc tgcacgagtc aggaacaagc ccgtccacag tggggtgagt 60
caagccccca cggcgggccg ggactgccac catcgtgcgg accccgcatc gccgcgggac 120
tcgggctgcc gtggctgctg gggagacctg gtgctgcagc cgctccggag ctctcggaaa 180
ctttcctccg cgctgtgcgc gggctgccta tcctttctgc tggcgctgct ggtgaggctg 240
gtccgcgggg aggtcggctg tgacctggag cagtgtaagg aggcggcggc ggcggaggag 300
gaggaagcag ccccgggagc agaaggggcc gtcttcccgg ggcctcgggg aggtgctccc 360
gggggcggtg cgcggctcag cccctggctg cagccctcgg cgctgctctt cagtctcctg 420
tgtgccttct tctggatggg cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct 480
gtcgcgctgc tggccgcctg ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc 540
ggggaggatc acttactctc actccccgcc gcgggggtgg tgctcagctg cttggccgcc 600
gcgacatggc tggtgctgag gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg 660
gtcaggaccg tgtccctcat ttccttagag aggttcaagg tcgcctggag accttacctg 720
gcgtacctgg ccggcgtgct ggggatcctc ttggccaggt acgtggaaca aatcttgccg 780
cagtccgcgg aggcggctcc aagggagcat ttggggtccc agctgattgc tgggaccaag 840
gaagatatcc cggtgtttaa gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg 900
tccggctgca gcagcaagtc ccatcggagg acctccctgc cctgtatacc gagggaacag 960
ctcatggggc attcagaatg ggaccacaaa cgagggccaa gaggatcaca gtcttcagga 1020
accagtatta ctgtggacat cgccgtcatg ggcgaagcca cggcctcatt accgacctcc 1080
tggcagaccc ttctcttcca ccaaacgtgt gccacatcct tgagagccgt gagcaacttg 1140
ctcagcacac agctcacctt ccaggccatt cacaagccca gagtgaatcc cgttacttcg 1200
ctcagtgaaa actatacctg ttctgactct gaagagagct ctgaaaaaga caagcttgct 1260
attccaaagc gcctgagaag gagtttgcct cctggcttgt tgagacgagt ttcttccact 1320
tggaccacca ccacctcggc cacaggtcta cccaccttgg agcctgcacc agtacggaga 1380
gaccgcagca ccagcatcaa actgcaggaa gcaccttcat ccagtcctga ttcttggaat 1440
aatccagtga tgatgaccct caccaaaagc agatccttta cttcatccta tgctatttct 1500
gcagctaacc atgtaaaggc taaaaagcaa agtcgaccag gtgccctcgc taaaatttca 1560
cctctttcat cgccctgctc ctcacctctc caagggactc ctgccagcag cctggtcagc 1620
aaaatttctg cagtgcagtt tccagaatct gctgacacaa ctgccaaaca aagcctaggt 1680
tctcacaggg ccttaactta cactcagagt gccccagacc tatcccctca aatcctgact 1740
ccacctgtta tatgtagcag ctgtggcaga ccatattccc aagggaatcc tgctgatgag 1800
cccctggaga gaagtggggt agccactcgg acaccaagtc gaacagatga cactgctcaa 1860
gttacctctg attatgaaac caataacaac agtgacagca gtgacattgt acagaatgaa 1920
gatgaaacag agtgcctgag agagcctctg aggaaagcat cggcttgcag cacctatgct 1980
cctgagacca tgatgtttct ggacaaacca attcttgctc ccgaacctct tgtcatggat 2040
aacctggact caattatgga gcagctaaat acttggaatt ttccaatttt tgatttagtg 2100
gaaaatatag gaagaaaatg tggccgtatt cttagtcagg tatcttacag actttttgaa 2160
gacatgggcc tctttgaagc ttttaaaatt ccaattaggg aatttatgaa ttattttcat 2220
gctttggaga ttggatatag ggatattcct tatcataaca gaatccatgc cactgatgtt 2280
ttacatgctg tttggtatct tactacacag cctattccag gcctctcaac tgtgattaat 2340
gatcatggtt caaccagtga ttcagattct gacagtggat ttacacatgg acatatggga 2400
tatgtattct caaaaacgta taatgtgaca gatgataaat acggatgtct gtctgggaat 2460
atccctgcct tggagttgat ggcgctgtat gtggctgcag ccatgcacga ttatgatcat 2520
ccaggaagga ctaatgcttt cctggttgca actagtgctc ctcaggcggt gctatataac 2580
gatcgttcag ttttggagaa tcatcacgca gctgctgcat ggaatctttt catgtcccgg 2640
ccagagtata acttcttaat taaccttgac catgtggaat ttaagcattt ccgtttcctt 2700
gtcattgaag caattttggc cactgacctg aagaaacact ttgacttcgt agccaaattt 2760
aatggcaagg taaatgatga tgttggaata gattggacca atgaaaatga tcgtctactg 2820
gtttgtcaaa tgtgtataaa gttggctgat atcaatggtc cagctaaatg taaagaactc 2880
catcttcagt ggacagatgg tattgtcaat gaattttatg aacagggtga tgaagaggcc 2940
agccttggat tacccataag ccccttcatg gatcgttctg ctcctcagct ggccaacctt 3000
caggaatcct tcatctctca cattgtgggg cctctgtgca actcctatga ttcagcagga 3060
ctaatgcctg gaaaatgggt ggaagacagc gatgagtcag gagatactga tgacccagaa 3120
gaagaggagg aagaagcacc agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct 3180
ccaaaaaaga agactttcaa aaggagaaaa atctactgcc aaataactca gcacctctta 3240
cagaaccaca agatgtggaa gaaagtcatt gaagaggagc aacggttggc aggcatagaa 3300
aatcaatccc tggaccagac ccctcagtcg cactcttcag aacagatcca ggctatcaag 3360
gaagaagaag aagagaaagg gaaaccaaga ggcgaggaga taccaaccca aaagccagac 3420
cagtgacaat ggatagaatg ggctgtgttt ccaaacagat tgacttgtca aagactctct 3480
tcaagccagc acaagcattt agatcacaac actgtagaaa tttgagatgg gcaaatggct 3540
attgcatttt gggattcttc gcattttgtg tgtatatttt tacagtgagg tacattgtta 3600
aaaacttttt gctcaaagaa gctttcacat tgcaacacca gcttctaagg attttttaag 3660
gagggaatat atatgtgtgt gtgtatataa gctcccacat agatacatgt aaaacatatt 3720
cacacccatg cacgcacaca catacacact gaaggccacg attgctggct ccacaattta 3780
gtaacattta tattaagata tatatatagt ggtcactgtg atataataaa tcataaagga 3840
aaccaaatca caaaggagat ggtgtggctt agcaaggaaa cagtgcagga aatgtaggtt 3900
accaactaag cagcttttgc tcttagtact gagggatgaa agttccagag cattatttga 3960
attctgatac atcctgccaa cactgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 4020
gtgtgtgaaa gagagacaga agggatggtt tgagaggggt cgcttgtgtg catgtgtgtg 4080
ctatatgtaa agagattttt gtggtttaag taactcagaa tagctgtagc aaatgactga 4140
atacatgtga acaaacagaa ggaagttcac tctggagtgt ctttgggagg caggctattc 4200
caaatgccct cgtcgattta gcttcaataa agggcctttt gctggtggag ggcactcaag 4260
ggctccctca gagggccacg tgtttggtat tacattactg ctatgcacca cttgaaggag 4320
ctctatcacc agcctgaaac ccgaagactg aggcattttc caggtctact tgcctaatga 4380
atgtatagga actgtctatg agtatggatg tcactcaact aagatcaaat caccatttaa 4440
gggggatggc attctttata cctaaacacc taagagctga agtcaggtct tttaatcagg 4500
ttagaattct aaatgatgcc agagaaggct tgggaaattg tacttcaggg tgatagcctg 4560
tgtcttctta atttactggg aaatatgtgg tagagaaagg aaagga 4606
gaggaagcag ccccgggagc agaaggggcc gtcttcccgg ggcctcgggg aggtgctccc 60
gggggcggtg cgcggctcag cccctggctg cagccctcgg cgctgctctt cagtctcctg 120
tgtgccttct tctggatggg cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct 180
gtcgcgctgc tggccgcctg ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc 240
ggggaggatc acttactctc actccccgcc gcgggggtgg tgctcagctg cttggccgcc 300
gcgacatggc tggtgctgag gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg 360
gtcaggaccg tgtccctcat ttccttagag aggttcaagg tcgcctggag accttacctg 420
gcgtacctgg ccggcgtgct ggggatcctc ttggccaggt acgtggaaca aatcttgccg 480
cagtccgcgg aggcggctcc aagggagcat ttggggtccc agctgattgc tgggaccaag 540
gaagatatcc cggtgtttaa gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg 600
tccggctgca gcagcaagtc ccatcggagg acctccctgc cctgtatacc gagggaacag 660
ctcatggggc attcagaatg ggaccacaaa cgagggccaa gaggatcaca gtcttcagga 720
accagtatta ctgtggacat cgccgtcatg ggcgaagcca cggcctcatt accgacctcc 780
tggcagaccc ttctcttcca ccaaacgtgt gccacatcct tgagagccgt gagcaacttg 840
ctcagcacac agctcacctt ccaggccatt cacaagccca gagtgaatcc cgttacttcg 900
ctcagtgaaa actatacctg ttctgactct gaagagagct ctgaaaaaga caagcttgct 960
attccaaagc gcctgagaag gagtttgcct cctggcttgt tgagacgagt ttcttccact 1020
tggaccacca ccacctcggc cacaggtcta cccaccttgg agcctgcacc agtacggaga 1080
gaccgcagca ccagcatcaa actgcaggaa gcaccttcat ccagtcctga ttcttggaat 1140
aatccagtga tgatgaccct caccaaaagc agatccttta cttcatccta tgctatttct 1200
gcagctaacc atgtaaaggc taaaaagcaa agtcgaccag gtgccctcgc taaaatttca 1260
cctctttcat cgccctgctc ctcacctctc caagggactc ctgccagcag cctggtcagc 1320
aaaatttctg cagtgcagtt tccagaatct gctgacacaa ctgccaaaca aagcctaggt 1380
tctcacaggg ccttaactta cactcagagt gccccagacc tatcccctca aatcctgact 1440
ccacctgtta tatgtagcag ctgtggcaga ccatattccc aagggaatcc tgctgatgag 1500
cccctggaga gaagtggggt agccactcgg acaccaagtc gaacagatga cactgctcaa 1560
gttacctctg attatgaaac caataacaac agtgacagca gtgacattgt acagaatgaa 1620
gatgaaacag agtgcctgag agagcctctg aggaaagcat cggcttgcag cacctatgct 1680
cctgagacca tgatgtttct ggacaaacca attcttgctc ccgaacctct tgtcatggat 1740
aacctggact caattatgga gcagctaaat acttggaatt ttccaatttt tgatttagtg 1800
gaaaatatag gaagaaaatg tggccgtatt cttagtcagg tatcttacag actttttgaa 1860
gacatgggcc tctttgaagc ttttaaaatt ccaattaggg aatttatgaa ttattttcat 1920
gctttggaga ttggatatag ggatattcct tatcataaca gaatccatgc cactgatgtt 1980
ttacatgctg tttggtatct tactacacag cctattccag gcctctcaac tgtgattaat 2040
gatcatggtt caaccagtga ttcagattct gacagtggat ttacacatgg acatatggga 2100
tatgtattct caaaaacgta taatgtgaca gatgataaat acggatgtct gtctgggaat 2160
atccctgcct tggagttgat ggcgctgtat gtggctgcag ccatgcacga ttatgatcat 2220
ccaggaagga ctaatgcttt cctggttgca actagtgctc ctcaggcggt gctatataac 2280
gatcgttcag ttttggagaa tcatcacgca gctgctgcat ggaatctttt catgtcccgg 2340
ccagagtata acttcttaat taaccttgac catgtggaat ttaagcattt ccgtttcctt 2400
gtcattgaag caattttggc cactgacctg aagaaacact ttgacttcgt agccaaattt 2460
aatggcaagg taaatgatga tgttggaata gattggacca atgaaaatga tcgtctactg 2520
gtttgtcaaa tgtgtataaa gttggctgat atcaatggtc cagctaaatg taaagaactc 2580
catcttcagt ggacagatgg tattgtcaat gaattttatg aacagggtga tgaagaggcc 2640
agccttggat tacccataag ccccttcatg gatcgttctg ctcctcagct ggccaacctt 2700
caggaatcct tcatctctca cattgtgggg cctctgtgca actcctatga ttcagcagga 2760
ctaatgcctg gaaaatgggt ggaagacagc gatgagtcag gagatactga tgacccagaa 2820
gaagaggagg aagaagcacc agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct 2880
ccaaaaaaga agactttcaa aaggagaaaa atctactgcc aaataactca gcacctctta 2940
cagaaccaca agatgtggaa gaaagtcatt gaagaggagc aacggttggc aggcatagaa 3000
aatcaatccc tggaccagac ccctcagtcg cactcttcag aacagatcca ggctatcaag 3060
gaagaagaag aagagaaagg gaaaccaaga ggcgaggaga taccaaccca aaagccagac 3120
cagtgacaat ggatagaatg ggctgtgttt ccaaacagat tgacttgtca aagactctct 3180
tcaagccagc acaagcattt agatcacaac actgtagaaa tttgagatgg gcaaatggct 3240
attgcatttt gggattcttc gcattttgtg tgtatatttt tacagtgagg tacattgtta 3300
aaaacttttt gctcaaagaa gctttcacat tgcaacacca gcttctaagg attttttaag 3360
gagggaatat atatgtgtgt gtgtatataa gctcccacat agatacatgt aaaacatatt 3420
cacacccatg cacgcacaca catacacact gaaggccacg attgctggct ccacaattta 3480
gtaacattta tattaagata tatatatagt ggtcactgtg atataataaa tcataaagga 3540
aaccaaatca caaaggagat ggtgtggctt agcaaggaaa cagtgcagga aatgtaggtt 3600
accaactaag cagcttttgc tcttagtact gagggatgaa agttccagag cattatttga 3660
attctgatac atcctgccaa cactgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3720
gtgtgtgaaa gagagacaga agggatggtt tgagaggggt cgcttgtgtg catgtgtgtg 3780
ctatatgtaa agagattttt gtggtttaag taactcagaa tagctgtagc aaatgactga 3840
atacatgtga acaaacagaa ggaagttcac tctggagtgt ctttgggagg caggctattc 3900
caaatgccct cgtcgattta gcttcaataa agggcctttt gctggtggag ggcactcaag 3960
ggctccctca gagggccacg tgtttggtat tacattactg ctatgcacca cttgaaggag 4020
ctctatcacc agcctgaaac ccgaagactg aggcattttc caggtctact tgcctaatga 4080
atgtatagga actgtctatg agtatggatg tcactcaact aagatcaaat caccatttaa 4140
gggggatggc attctttata cctaaacacc taagagctga agtcaggtct tttaatcagg 4200
ttagaattct aaatgatgcc agagaaggct tgggaaattg tacttcaggg tgatagcctg 4260
tgtcttctta atttactggg aaatatgtgg tagagaaagg aaagga 4306
tgatcgtttc tgcccgtgct tgttttc 27
cagggaaagt ttccgagagc tccggag 27
ctagatccca gggaacatca atagagttta agtccattga acagatactg aattcttttt 60
cataatctgc caaaaaaagg ttagcttgaa aattttcttt tagtttctca aatatcacac 120
tgctgcagta cacgaacctt tactcattaa taactaaggt cctgattttt ttcatatgct 180
ttgctcgaag atgtagtatt ttgcagccat agacagtctt ctaagatctc tcctagtgtt 240
aaccacctat gctcacctct cccttgagat ttttctttat tttttgatga actatctggg 300
cttttaaact ttgttaacct tttttgagga tacggtcact taatctcaat gtaattttac 360
tttccacagt caaaaactat tgtgaatact catgcactgg atttaaatga ctgctgcctc 420
tccttccttt ctttttatac tattgtggtc taggtaaggc tgattcttcc atcatttgaa 480
ccaacaggcc aggcttgggt tctcataaag cagaccttcc agcaggagcg accaaaggat 540
gacactgtca cctgaaattg gactgctgtt gtacctgact tgggaacatc tttgaatcag 600
acagtagaag tggctgtcat tttcagggac agtagaaagt atgttggctc tcatctgcca 660
agtaggcaaa cacaatcttt tttttttttt tttccttcca acgttctagg gagctcagcc 720
tcagggctag ccgcagcccc ccacaccccg gggctgcggt gggctgcgcg gtggatcaac 780
ctcagcagcc cctgctccag cctgtagggt gaaccggccg ctttcccagc aaaggagcaa 840
tcgagctgag ggtagcgcct cctccgcagg agggggcggg agctcggctg agaaagcttt 900
cctagggagt tgccttaaag aaagaaagcg gaattgtcga tcactccagt tgccagtttt 960
atacaatttt aagcagtcgt cgccactcgt ttcccctttg caaaactgca aatcaccacc 1020
aaccttgcat caaatagaag tggggaggga aaaaaaaagc aaatctcctt ctcccttctc 1080
accctccctt tcttctcacc ctcccttcct ctcttactcg ctccttctcc ctccctccct 1140
tctgcggctg ccgctagtct ctcggtcctg gctctctctc cgacgggact tagcaacttc 1200
ttatttctca gccccttgtc attttttttt ttccatcctt tgccatgaat tggattgaca 1260
gaggcggggg aggctttgct ttctagccca gggaatggcg atcgcgtcct ggggccgtgc 1320
ggggagaacg gcagaggaga aagaaagagt gatagaaaaa gagctgcagg aaggaggaga 1380
aggagacctc catctacctg cgggcccggc gcgctgcagc gcacgcagcg cgacgtgcgc 1440
ctcggaatgg cccggagccc gccctgcgcc ccggctcctc cagcgtcagc ggctcctgcg 1500
cgcgggatgc attgggcaat ttttgaaatc ctgaagtagg aagagacccc ggaggataga 1560
agtcgggggt gggggtggag cagagaatct gtgaaagata ttcaaagaga aaaggggaat 1620
cctgatcctt tctgcccgtg cttgttttca acttgagcgt gctagccttt aacttgaaga 1680
agtctcattg gagcatctag cattctccag gagttattcg aaagctgaaa ctttcagtgg 1740
attgtgggcc tggggagaag aaggattccg agggtggaat tgggaagagc gtgcgtgcgt 1800
gtgtgtgtgt gtgtgtgtgc gcgcgcgcgt gggtcggggc gggggcgtcg gggggccact 1860
gggaattcag tgaagagggc accctatacc 1890