Cyclic nucleotide analogs as biochemical tools and prospective drugs

Pharmacology & Therapeutics 87 (2000) 199–226

0163-7258/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.PII:

S0163-7258(00)00051-6

Associate editor: D. Shugar

Cyclic nucleotide analogs as biochemical tools and prospective drugs

Frank Schwede

a

, Erik Maronde

b

, Hans-Gottfried Genieser

c

, Bernd Jastorff

a,

*

a

Center for Environmental Research and Environmental Technology, Department of Bioorganic Chemistry, University of Bremen,Leobener Straße, D-28359 Bremen, Germany

b

Institute for Peptide Research, Feodor-Lynen-Straße 31, D-30625 Hannover, Germany

c

BIOLOG Life Science Institute, P.O. Box 107125, D-28071 Bremen, Germany

Abstract

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealthof synthetic analogs of cAMP and cGMP, only a few have been explored with regard to their therapeutic potential. Some of the first-gen-eration cyclic nucleotide analogs were promising enough to be tested as drugs, for instance

N

6

,

O

2

9

-dibutyryl-cAMP and 8-chloro-cAMP(currently in clinical Phase II trials as an anticancer agent). Moreover, 8-bromo and dibutyryl analogs of cAMP and cGMP have becomestandard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers ofadenosine 3

9

,5

9

-cyclic monophosphorothioate and guanosine 3

9

,5

9

-cyclic monophosphorothioate as competitive inhibitors of cAMP- andcGMP-dependent protein kinases, as well as subsequent development of related analogs, has proven very useful for studying the molecu-lar basis of signal transduction. These analogs exhibit a higher membrane permeability, increased resistance against degradation, and im-proved target specificity. Furthermore, better understanding of signaling pathways and ligand/protein interactions has led to new thera-peutic strategies. For instance, Rp-8-bromo-adenosine 3

9

,5

9

-cyclic monophosphorothioate is employed against diseases of the immunesystem. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research, butalso highlights some historical findings in the field. © 2000 Elsevier Science Inc. All rights reserved.

Keywords:

Cyclic nucleotide analogs; cAMP-dependent protein kinase; cGMP-dependent protein kinase; 8-Chloro cyclic AMP; Clinical studies

Abbreviations:

AC, adenylate cyclase; AM, acetoxymethyl; 8-Br-cAMP, 8-bromo-cyclic AMP; 8-Br-cGMP, 8-bromo-cyclic GMP; Bt

2

-cAMP,

N

6

,

O

2

9

-dibu-tyryl-cyclic AMP; Bt

2

-cAMP/AM, Bt

2

-cAMP acetoxymethyl ester; Bt

2

-cGMP,

N

2

,

O

2

9

-dibutyryl-cyclic GMP; C, catalytic; cAMP, cyclic AMP; cGMP, cyclicGMP; 8-Cl-cAMP, 8-chloro-cAMP; CNG, cyclic nucleotide-gated; cNMP, cyclic nucleotide monophosphate; CREB, cyclic AMP responsive element bind-ing; CVI, common variable immunodeficiency; Epac, exchange protein directly activated by cyclic AMP; GC, guanylate cyclase; GEF, guanine nucleotideexchange factor; GLP, glucagon-like peptide; HCN, hyperpolarization-activated, cyclic nucleotide-gated; HIV, human immunodeficiency virus; ICER,inducible cyclic AMP early repressor; IL, interleukin; 6-MBC-cAMP,

N

6

-mono-

tert

-butylcarbamoyl-cyclic AMP; MDR, multidrug resistance;

N

2

-Bt-cGMP,

N

2

-monobutyryl-cyclic GMP;

N

6

-Bt-cAMP,

N

6

-monobutyryl-cyclic AMP; NK, natural killer; NO, nitric oxide; 8-pCPT-cAMP, 8-

para

-chlorophenylthio-cyclic AMP; 8-pCPT-cGMP, 8-

para

-chlorophenylthio-cyclic GMP; PDE, phosphodiesterase; PET-cGMP,

b

-phenyl-1,

N

2

-etheno-cyclic GMP; PKA, proteinkinase A; PKA-I, protein kinase A Type I; PKA-II, protein kinase A Type II; PKG, protein kinase G; PVR, pulmonary vascular resistance; R, regulatory;Rp-8-Br-cAMPS, Rp-8-bromo-adenosine 3

9

,5

9

-cyclic monophosphorothioate; Rp-8-Br-PET-cGMPS, Rp-8-bromo-

b

-phenyl-1,

N

2

-etheno-guanosine 3

9

,5

9

-cyclic monophosphorothioate; Rp-cAMPS, Rp-diastereomer of adenosine 3

9

,5

9

-cyclic monophosphorothioate; Rp-cGMPS, Rp-diastereomer of guanosine3

9

,5

9

-cyclic monophosphorothioate; Sp-5,6-Cl

2

-cBIMPS, Sp-5,6-dichloro-1-

b

-

D

-ribofuranosyl-benzimidazole-3

9

,5

9

- cyclic monophosphorothioate; Sp-8-Br-PET-cGMPS, Sp-8-bromo-

b

-phenyl-1,

N

2

-etheno-guanosine 3

9

,5

9

-cyclic monophosphorothioate; Sp-cAMPS, Sp-diastereomer of adenosine 3

9

,5

9

-cyclicmonophosphorothioate; Sp-cGMPS, Sp-diastereomer of guanosine 3

9

,5

9

-cyclic monophosphorothioate; ST

a

, heat-stable enterotoxin from

Escherichia coli

;

TCR, T-cell receptor.

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2002. Receptor proteins of cyclic AMP and cyclic GMP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

2.1. Proteins involved in formation and degradation of cyclic AMP and cyclic GMP. . . 2012.2. Target proteins of cyclic AMP and cyclic GMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2012.3. Crosstalk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

3. Cyclic nucleotide analogs as biochemical tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2033.1. Properties of traditional cyclic nucleotide analogs . . . . . . . . . . . . . . . . . . . . . . . . . . 203

3.1.1.

N

6

,

O

2

9

-Dibutyryl-cyclic AMP and

N

2

,

O

2

9

-dibutyryl-cyclic GMP . . . . . . . . 204

* Corresponding author. Tel.:

1

49-421-218-7644; fax:

1

49-421-218-7645.

E-mail address

: [email protected] (B. Jastorff); [email protected] (E. Maronde); [email protected] (H.-G. Genieser); [email protected](F. Schwede).

200

F. Schwede et al. / Pharmacology & Therapeutics 87 (2000) 199–226

3.1.2. 8-Bromo-cyclic AMP and 8-bromo-cyclic GMP. . . . . . . . . . . . . . . . . . . . . 2053.2. Mapping studies as a key for improved cyclic nucleotide analogs . . . . . . . . . . . . . . 205

3.2.1. Cyclic AMP-dependent protein kinase Type I and Type II . . . . . . . . . . . . . 2053.2.2. Cyclic GMP-dependent protein kinase Type I

a

, Type I

b

, and Type II . . . . 2063.2.3. Mapping studies with testkit analogs and the discovery of

Rp-adenosine 3

9

,5

9

-cyclic monophosphorothioate. . . . . . . . . . . . . . . . . . . . 207 3.3. Rp- and Sp-adenosine 3

9

,5

9

-cyclic monophosphorothioate analogs as newbiochemical tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

3.4. Rp- and Sp-guanosine 3

9

,5

9

-cyclic monophosphorothioate analogs as newbiochemical tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

3.5. Bioactivatable, membrane-permeant prodrugs of cyclic nucleotides . . . . . . . . . . . . 2104. Cyclic nucleotide analogs as prospective drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

4.1. 8-Chloro-cyclic AMP, a new anticancer drug? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2114.2. Other cyclic nucleotide analogs and growth inhibition . . . . . . . . . . . . . . . . . . . . . . . 2144.3. Cyclic nucleotide analogs in organ transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . 2144.4. Pulmonary hypertension or asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2164.5. Rp-8-bromo-adenosine 3

9

,5

9

-cyclic monophosphorothioate, a new leadstructure against diseases of the immune system? . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

1. Introduction

Cyclic AMP (cAMP) and cyclic GMP (cGMP) (see Fig. 1)act as second messengers in a multitude of cellular processes,such as gene control, chemotaxis, proliferation, differentiation,and apoptosis. Several regulatory diseases are associatedwith unusually high or low levels of cAMP and/or cGMP.Between 1960 and 1980, hundreds of analogs of cAMP andcGMP have been synthesized and screened for their therapeuticpotential, especially against diabetes, asthma, cardiovasculardiseases, or as anticancer agents (Posternak et al., 1962;Miller & Robins, 1976; Miller, 1977). For instance, cAMP iswell known to be involved in the control of cell differentiationand proliferation in cancer (Ryan & Heidrick, 1974; Sheppard,1974; Boynton & Whitfield, 1983). Several early studiesreported an inhibitory effect of cAMP analogs on cell prolif-eration (Pastan et al., 1975; Friedman, 1976). Experimentswith animals have demonstrated that cAMP analogs inhibittumor growth in vivo (Cho-Chung, 1979a, 1979b). However,lack of tissue specificity, metabolic instability, undesiredside effects and insufficient cell penetration are major limitingfactors for most of these so-called first-generation analogs(Pastan et al., 1975; Friedman, 1976; Boynton & Whitfield,1983). Only a few of these have been further tested as potentialtherapeutics, especially 8-chloro-cAMP (8-Cl-cAMP), whichrecently entered Phase II clinical trials as an anticancer agent.

In addition, the 8-bromo and dibutyryl analogs of cAMPand cGMP are considered standard tools in current signaltransduction research, and for some clinical applications, suchas organ transplantation (e.g., Kayano et al., 1999) and asthma(Lawson et al., 1995). This review focuses critically on thebenefits and potential problems that may arise when the mostpopular cyclic nucleotide analogs, namely,

N

6

,

O

2

9

-dibutyryl-cAMP (Bt

2

-cAMP),

N

2

,

O

2

9

-dibutyryl-cGMP (Bt

2

-cGMP),8-bromo-cAMP (8-Br-cAMP), and 8-bromo-cGMP (8-Br-

cGMP) are used as biochemical tools. Moreover, new strategiesfor isozyme-selective activation of either cAMP- or cGMP-dependent protein kinases (PKAs, PKGs) are discussed.Although no specific activator on the basis of a single cyclic nu-cleotide analog has been described yet for any of theseisozymes, considerable work has been done on the developmentof PKA isozyme-directed pairs of cyclic nucleotide analogs(Øgreid et al., 1985; Døskeland et al., 1991, 1993), an approachuseful for identification of specific PKA isozymes involved inseveral physiological processes (Sections 3.2.1 and 4.5).

In the PKG field, some cGMP analogs with high in vitroactivation potencies and useful properties for experimentswith intact cells were identified or developed recently (e.g.,Sekhar et al., 1992; Pöhler et al., 1995). Results with puri-fied enzyme preparations have led to a new strategy foridentification of PKG isozymes involved in physiologicalprocesses (Vaandrager et al., 1997).

We focus on a new second generation of hydrolysis-resis-tant Sp- and Rp-adenosine 3

9

,5

9

-cyclic monophosphorothio-ate (cAMPS) and -guanosine 3

9

,5

9

-cyclic monophospho-rothioate (cGMPS) analogs with agonistic or antagonistic

Fig. 1. Chemical structures of cAMP and cGMP.

F. Schwede et al. / Pharmacology & Therapeutics 87 (2000) 199–226

201

properties compared with cAMP and cGMP. After the dis-covery that Rp-cAMPS and Rp-cGMPS are competitive inhib-itors for PKA and PKG, respectively (De Wit et al., 1982; Ro-thermel et al., 1983; Butt et al., 1990), several new derivativesof these parent compounds have been synthesized and tested.Some of these, for instance, Sp-5,6-dichloro-1-

b

-

D

-ribo-furanosyl-benzimidazole-3

9

,5

9

-cyclic monophosphorothioate(Sp-5,6-Cl

2

-cBIMPS), Sp-8-bromo-

b

-phenyl-1,

N

2

-etheno-cGMPS (Sp-8-Br-PET-cGMPS), as well as Rp-8-bromo-cAMPS (Rp-8-Br-cAMPS) and Rp-8-bromo-

b

-phenyl-1,

N

2

-etheno-cGMPS (Rp-8-Br-PET-cGMPS), possess excellentproperties for in vitro studies (e.g., Sandberg et al., 1991;Butt et al., 1995b; Gjertsen et al., 1995). Moreover, for Rp-8-Br-cAMPS, initial results of possible clinical relevance fordiseases of the immune system have been reported (Aan-dahl et al., 1998; Aukrust et al., 1999).

Some recent work concerning bioactivatable, highlymembrane-permeant cyclic nucleotide prodrugs is pre-sented, and some of the biological activities of these third-generation cyclic nucleotide monophosphate (cNMP) drugsare reported and compared with the activity of the parentcompounds and currently established cNMP analogs.

2. Receptor proteins of cyclic AMP and cyclic GMP

2.1. Proteins involved in formation and degradation of cyclic AMP and cyclic GMP

The development of cAMP and cGMP analogs as thera-peutics or tools in signal transduction research calls foridentification and characterization of cyclic nucleotide tar-get proteins, as well as proteins involved in the anabolismand catabolism of cyclic nucleotides. The intracellular lev-els of cAMP and cGMP are determined by a balance be-tween formation by adenylate and guanylate cyclases (AC,GC), respectively, and degradation by cyclic nucleotidephosphodiesterases (PDEs). The AC family consists of atleast nine membrane-associated isoforms (AC1–AC9) acti-vated by G-proteins, and represents the major pathway togenerate increased cAMP levels in intact cells or tissues,since extracellular cAMP is considered impermeable to thecell membrane (see Cooper et al., 1998; Taussig & Zimmer-mann, 1998; Simonds, 1999; Hurley, 1999). In contrast tothe AC isozymes, GCs are divided into soluble forms (GC-S

a

1–3

b

1–3

), predominantly activated by nitric oxide (NO),and membrane-receptor forms (GC-A to GC-G, retGC).The latter receptors are either activated by natriuretic pep-tides, such as atrial natriuretic peptide, brain-type natriureticpeptide, natriuretic peptide type C, the heat-stable entero-toxin from

Escherichia coli

(ST

a

), and the intestinal peptideguanylin, or possess no known ligand (see Schmidt et al.,1993; Murad, 1994; Leitman et al., 1994; Forssmann et al.,1998; Koesling & Friebe, 1999). To our knowledge, PDEsare the only known enzymes responsible for the intracellu-lar degradation of cAMP and cGMP to their correspondingmonophosphates 5

9

-AMP and 5

9

-GMP, the first step in the

purine salvage pathway (Thompson, 1991). The PDE super-family in mammals is divided into at least 10 functionallydifferent isozyme classes (PDE1–PDE10; Corbin & Fran-cis, 1999). Most PDE families consist of more than oneunique gene product, and multiple splice-variants were de-scribed, leading to more than 30 members (see Burns et al.,1996; Corbin & Francis, 1999; Juilfs et al., 1999). ThecGMP-stimulated PDE2 and the cGMP-inhibited PDE3preferentially hydrolyze cAMP. In addition, they are regu-lated by cGMP through a competitive (PDE3) or allosteric(PDE2) mechanism. Therefore, these two isozyme familiesrepresent excellent targets for crosstalk between cAMP andcGMP signaling (Section 2.3). PDE4, PDE7, and PDE8 arehighly specific for degradation of cAMP, while PDE5,PDE6, and PDE9 are cGMP-specific. Like PDE2, PDE5,PDE6, and PDE10 exhibit both noncatalytic and catalyticbinding sites for cGMP (Corbin & Francis, 1999).

2.2. Target proteins of cyclic AMP and cyclic GMP

PKAs constitute an ubiquitous enzyme family present inall eukaryotic cells, responsible for the mediation of mostbiological effects of cAMP (Taylor et al., 1990; Francis &Corbin, 1994a). Therefore, up to now, PKAs represent themain target enzymes for the development of cyclic nucle-otide analogs that might serve as future drugs or tools forbiochemical and pharmacological studies. PKA exists as aninactive holoenzyme complex of a dimeric regulatory (R

2

) sub-unit and two monomeric catalytic (2 C) subunits in the ab-sence of cAMP. Two major forms of PKA, Type I and TypeII (PKA-I and PKA-II), have been identified, determined bytheir different R-subunits (RI and RII). The R-subunits arefurther subclassified into

a

- and

b

-forms, leading to fourdistinct R-subunits, RI

a

, RI

b

, RII

a

, and RII

b

, which are allencoded by separate genes (Rubin, 1994; Taskén et al.,1995). Each R-subunit consists of two kinetically differentbinding domains for cAMP, known as site A (or site I; orrapid site) and site B (or site II; or slow site), arranged intandem (Døskeland, 1978; Øgreid & Døskeland, 1980;Øgreid et al., 1983). Additionally, three isoforms of theC-subunit of PKA, with high sequence homologies, havebeen described (C

a

, C

b

, and C

g

), and the human X chro-mosome-encoded protein kinase X was recently identifiedas a novel C-subunit of PKA-I (Zimmermann et al., 1999).The diversity of PKA and its subunit composition, as wellas its properties, have been extensively reviewed elsewhere(McKnight, 1991; Døskeland et al., 1993; Francis &Corbin, 1994a). Activation of PKA holoenzyme depends onbinding of two molecules of cAMP to each R-subunit andsubsequent dissociation of the C-subunits. After liberation,the active free C-subunit can phosphorylate target proteins.This results in specific cellular responses initiated by an ex-tracellular first messenger (hormone, neurotransmitter, etc.)and mediated by cAMP as second messenger. For this rea-son, cAMP and PKA are involved in a multitude of cellularprocesses, for instance, metabolism, differentiation, prolif-

202 F. Schwede et al. / Pharmacology & Therapeutics 87 (2000) 199–226

eration, apoptosis, gene transcription, ion flux, neurotransmit-ter release, or long-term potentiation (Cho-Chung & Clair,1993; Francis & Corbin, 1994a; Gjertsen & Døskeland, 1995;Qi et al., 1996; Montminy, 1997; Nguyen & Kandel, 1997).

cAMP is the natural second messenger for olfactorytransduction by modulating a cyclic nucleotide-gated (CNG)cation channel in the olfactory epithelium (Firestein, 1992;Zufall et al., 1994), although cGMP is reported to activatethis channel with similar potency (Nakamura & Gold, 1987;Goulding et al., 1994).

A new class of hyperpolarization-activated cation chan-nels, which are additionally modulated by cAMP (and to alesser extent by cGMP), have been cloned recently from seaurchin and mouse (Gauss et al., 1998; Ludwig et al., 1998;Santoro et al., 1998). After activation by hyperpolarization,the initial kinetics are prolonged by direct binding of cAMPor cGMP to a cyclic nucleotide binding domain highly simi-lar to CNG channels and PKA. Since the use of differentnames for the same type of proteins can produce misunder-standings, the term HCN (Hyperpolarization-activated, Cy-clic Nucleotide-gated) for this type of channels was proposedrecently (Clapham, 1998; Biel et al., 1999a). HCN channelsshare several characteristics with the already describedmixed Na1/K1 currents, named Ih or If channels, and thus,seem to be at least members of the same family of proteins(DiFrancesco, 1993; Pape, 1996; Biel et al., 1999a; Ludwiget al., 1999). These channels are thought to have severalphysiological functions; most prominent is their pacemakeractivity in heart cells and spontaneously active neurons.

In late 1998, three members of another new family ofcAMP-binding proteins were identified by two differentgroups (De Rooij et al., 1998; Kawasaki et al., 1998). Theseproteins are regulated by cAMP and exhibit a single cAMP-binding domain with close sequence similarities to PKA-Iand -II, and, in addition, a guanine nucleotide exchange factor(GEF) domain. Up to now, two different names for thisenzyme family have been in use, Epac (exchange proteindirectly activated by cAMP) and cAMP-GEF-I and -II.cAMP-GEF-I and Epac are widely expressed in human tissues,while cAMP-GEF-II is detected predominantly in adrenalgland and brain (De Rooij et al., 1998; Kawasaki et al., 1998).Upon binding of cAMP, Epac and cAMP-GEF demonstrateGEF activity towards Rap1, a small Ras-like GTPase, whichis suggested to be a negative regulator of Ras by suppressingoncogenic cell transformation (Kitayama et al., 1989). Fur-thermore, Rap1 is reported to have a function in platelet ag-gregation (Franke et al., 1997), cell differentiation (York etal., 1998), cell proliferation (Altschuler & Ribeiro-Neto,1998), and T-cell anergy (Boussiotis et al., 1997). These find-ings strongly suggest a PKA-independent direct coupling be-tween cAMP- and Ras superfamily-signaling.

Although assessments of the functions of HCN channelsand Epac/cAMP-GEF proteins in cellular processes are ea-gerly awaited, it appears important to reformulate and tochallenge the dogma that cAMP exerts its physiologicalfunctions almost solely through activation of PKA.

Other receptor proteins were found in bacteria, wherecAMP activates directly the catabolite gene activator-pro-tein, leading to gene transcription (Peterkowski, 1976), andin the slime mold Dictyostelium discoideum, where cAMPcan act as a chemotactic first messenger through binding tocell surface receptors (Klein et al., 1988; Saxe et al., 1991).

cGMP is able to regulate numerous receptor proteins,namely PKGs, CNG ion channels, and several PDEs. Fol-lowing the first descriptions of cGMP-dependent kinase ac-tivity in arthropods (Kuo & Greengard, 1970) and mamma-lian tissue (Hofmann & Sold, 1972), so far three differentmammalian PKGs (Types Ia, Ib, and II) have been isolated,characterized, and expressed as recombinant proteins (DeJonge, 1981; Lincoln et al., 1988; Wolfe et al., 1989; Ruthet al., 1991; Uhler, 1993; Jarchau et al., 1994; Meinecke etal., 1994). All PKG isozymes currently are accepted to behomodimeric proteins, although PKG-II previously was de-scribed as a monomer (De Jonge, 1981). This is now be-lieved to be an artefact, most likely produced by a specificendogenous protease during purification (Vaandrager et al.,1997). PKG-I isozymes are present in relatively high con-centrations in smooth muscle, lung, heart, platelets, cerebel-lum, hippocampus, kidney, and growth plate chondrocytes,and are predominantly cytosolic (see Francis & Corbin,1994b; Lohmann et al., 1997; Eigenthaler et al., 1999; Pfei-fer et al., 1999). PKG-Ia and -Ib exhibit the highest aminoacid sequence homologies, with differences only in theamino termini, leading to the assumption that both enzymesarise from an alternative mRNA splicing event of a single-gene product (Francis & Corbin, 1994b). PKG-II was firstidentified in rat and pig intestinal microvilli, but has nowbeen detected also in kidney, several regions of the brain,and bone (Pfeifer et al., 1999). Endogenous PKG-II is de-scribed as a membrane-associated enzyme, while differentrecombinant PKG-II proteins are characterized as soluble(recombinant mouse brain PKG-II) or membrane-associated(recombinant rat intestine PKG-II), perhaps dependent ondifferent N-terminal modifications (Gamm et al., 1995;Vaandrager et al., 1997). Each monomer of the kinases con-tains two cGMP-binding sites arranged in tandem and thecatalytic phosphotransferase domain on the same polypep-tide. Therefore, PKGs are not dissociated in their activatedform after binding of four molecules of cGMP, in strikingcontrast to PKA. PKG-I isozymes are involved in a multi-tude of physiological functions; for instance, smooth musclerelaxation, inhibition of platelet aggregation, inhibition ofendothelial cell permeability, and reduction of cardiac myo-cyte contractility. These functions provide considerable evi-dence that PKG-I is an important modulator of the cardio-vascular system, counteracting hypertension, thrombosis orvascular remodeling, and atherosclerosis (see Eigenthaler etal., 1999; Pfeifer et al., 1999). This physiological signifi-cance of PKG-I is further illustrated by the reported hyper-tension of knock-out mice lacking both PKG-I isoforms(Pfeifer et al., 1998). Additional studies on the functionalrole of PKG-I in the brain (including long-term depression

F. Schwede et al. / Pharmacology & Therapeutics 87 (2000) 199–226 203

and long-term potentiation; Aitken et al., 1981; Tsou et al.,1993; Zhuo et al., 1994) and the recently described regulationof gene expression (including cell growth and differentiation;Gudi et al., 1996; Belsham et al., 1996; Collins & Uhler,1999) are eagerly awaited to provide an improved under-standing of PKG action (see Eigenthaler et al., 1999; Pfeiferet al., 1999). PKG-II is concluded to mediate the effects ofSTas and guanylin on chloride secretion in intestinal mucosaby phosphorylation of the cystic fibrosis transmembrane con-ductance regulator protein (Pfeifer et al., 1996; Lohmann etal., 1997; Vaandrager et al., 1997), as well as control of bonegrowth (Pfeifer et al., 1996). The role of PKG-II in the kidneyand the brain has to be explored and distinguished from PKG-Iin more detail; however, it is possible that PKG-II mediates atleast some of the cGMP effects on the renin/angiotensin sys-tem in the kidney, as well as effects of NO and increasedcGMP levels in the brain (Hawkins et al., 1994; Gage et al.,1997; Lev-Ram et al., 1997; Lohmann et al., 1997; Eigentha-ler et al., 1999; Pfeifer et al., 1999).

CNG channels, with cGMP as the intrinsic effector mol-ecule, were first identified in rod and cone photoreceptors,both exerting a central role in the visual process (Fesenko etal., 1985; Yau & Nakatani, 1985; Cobbs et al., 1985; Haynes& Yau, 1985; Baylor, 1996). In the dark, cGMP-binding toCNG channels keeps the channels in their open state. Lightinduces hydrolysis of cGMP by the rhodopsin/transducin/PDE6-cascade, leading to channel closure and membranehyperpolarization, which is the physiological response ofphotoreceptors (Palczewski, 1994). Currently, five differentmembers of CNG channels (CNG1–5) with high sequencehomologies have been described in mammals (see Biel etal., 1999b, 1999c), detected not only in sensory cells, butalso in the CNS and various peripheral tissues, includingheart, colon, aorta, and kidney (Distler et al., 1994; Ding, C.et al., 1997). They may be involved in several processes,such as hormone synthesis and secretion in the pineal gland(Dryer & Henderson, 1991; Bönigk et al., 1996; Sautter et al.,1997), development and normal function of the mammalianbrain (Zufall et al., 1997), control of chemotactic movementof sperm cells (Weyand et al., 1994), and regulation of syn-aptic efficiency in CA1–3 hippocampal neurons (Chen &Schofield, 1995; Zufall et al., 1997). The functional role ofCNG channels in most nonsensory tissues is not clear yet,but due to their good permeability for Ca21 ions comparedwith other physiologically relevant cations, it appears likelythat these proteins represent cGMP-operated Ca21 channels(Biel et al., 1999b, 1999c) and, therefore, are an additionalimportant link to modulate the Ca21 signaling pathway.

2.3. Crosstalk

Several lines of evidence indicate that the cAMP andcGMP second messenger pathways function not only asseparated mechanisms, but can interfere vice versa at sev-eral target proteins. Under certain conditions, cytosolic GCcan synthesize, besides cGMP, also cAMP (5–15% the rate

of cGMP formation; Mittal et al., 1979). Although PKA andPKG isozymes are activated predominantly by their naturaleffector cyclic nucleotides, it is likely that in certain situa-tions, large intracellular increases of one of the second mes-sengers can result in cross-activation. For instance, cGMP isreported to inhibit platelet-derived growth factor-evokedproliferation of cultured smooth muscle cells (Cornwell etal., 1994) and to stimulate intestinal Cl2 transport (Forte etal., 1992; Chao et al., 1994) by activation of PKA. cAMPcan activate PKG and mediate smooth muscle relaxation inpig coronary arteries (Jiang et al., 1992) or phosphorylatethe inositol 1,4,5-trisphosphate receptor in intact rat aorta(Komalavilas & Lincoln, 1996). Both cAMP and cGMP canmodulate Ca21 channels in rabbit portal vein by activationof the opposing protein kinase (Ruiz-Velasco et al., 1998).In some tissues or cells, such as heart, adrenal gland, plate-lets, and hippocampal neurons, activation of cGMP-stimu-lated PDE2 is an accepted or proposed mechanism ofcGMP-controlled regulation of cAMP hydrolysis (Doerner& Alger, 1988; MacFarland et al., 1991; Sonnenburg &Beavo, 1994; Dickinson et al., 1997). In platelets, increasedlevels of cGMP can inhibit aggregation through at least twomechanisms, activation of PKG-I and inhibition of PDE3,the major PDE isoform in platelets. The latter event is com-bined with elevated levels of cAMP, which itself can coun-teract platelet activation (Maurice & Haslam, 1990; Bowen& Haslam, 1991). Cardiac contractility is assumed to beregulated by a sensitive balance of cAMP and cGMP levelsand activities of PDE2 and PDE3, especially for regulationof the basal calcium current (RivetBastide et al., 1997).

Whether cross-activation between cAMP and cGMP path-ways is rare or commonplace in cellular signal transduction isnot established, but one may speculate that crosstalk leads to afine regulation between these two second-messenger systems.

3. Cyclic nucleotide analogs as biochemical tools

3.1. Properties of traditional cyclic nucleotide analogs

The multitude of well-known “old” target proteins forcyclic nucleotides, such as PDEs, cNMP-regulated proteinkinases, and ion channels, in combination with the recentlyidentified “new” cNMP-binding proteins, and the emergingknowledge about potential crosstalk at several steps of thecAMP and cGMP signaling cascades, represents an enor-mous target complexity (Sections 2.1 and 2.2). So far, noneof the common cyclic nucleotide analogs has been testedwith every isozyme involved in cAMP and cGMP signaling.Therefore, the important feature of analog specificity, dis-cussed on the basis of currently available data in this sec-tion, is considered preliminary, especially with respect tothe “new” cNMP-binding proteins and crosstalk. Despitethis target complexity, several cNMP analogs have shownexcellent properties as tools in signal transduction researchin vitro and in vivo, and even in some important clinical ap-plications (Section 4).


Since cAMP and cGMP are almost unable to penetrateintact cellular membranes due to the polar ionic interactionpotential of the cyclic phosphate moieties (Fig. 1, Table 1),several hundred cyclic nucleotide analogs with hydrophobicsubstituents have been synthesized and widely used to elu-cidate the functional role of the cAMP and cGMP signalcascades in biological systems (Posternak et al., 1962;Miller & Robins, 1976; Miller, 1977). Four of these,namely, Bt2-cAMP, Bt2-cGMP, 8-Br-cAMP, and 8-Br-cGMP (Fig. 2), have received major attention and arewidely used tools for testing the role of cAMP/cGMP andPKA/PKG in biological processes.

3.1.1. N6,O29-Dibutyryl-cyclic AMP andN2,O29-dibutyryl-cyclic GMP

Bt2-cAMP and Bt2-cGMP, as lipophilic analogs ofcAMP and cGMP, possess considerable membrane perme-ability (Braumann & Jastorff, 1985; Krass et al., 1997; Ta-ble 1), one of several required properties of cyclic nucle-otides for use in cell culture. However, after passivediffusion into the cell, the dibutyryl analogs are still inactivetowards PKA and PKG, since the 29-hydroxy group, whichis crucial for binding and activation, is blocked by one ofthe butyrate substituents (Jastorff et al., 1979; Øgreid et al.,1985; Corbin et al., 1986). Bioactivation of the compoundsoccurs through intracellular hydrolysis of the 29-butyrate byendogenous nonspecific esterases or amidases, leading toN6-monobutyryl-cAMP (N6-Bt-cAMP) and N2-monobu-tyryl-cGMP (N2-Bt-cGMP). N6-Bt-cAMP is a potent activa-tor of both PKA-I and -II, with Kas of 76 nM and 229 nM,respectively (Øgreid et al., 1985), while PKG-Ia is acti-

vated half-maximally at 34 mM (Corbin et al., 1986). Forthis reason, N6-Bt-cAMP is considered to be an almost se-lective activator of PKA versus PKG, although to ourknowledge, PKG-Ib and -II have not been tested. N2-Bt-cGMP is only a poor stimulator of PKG-Ia, with a Ka of 2mM compared with cGMP (Ka of 110 nM; Corbin et al.,1986), and in general, is ineffective when used with intactcell preparations (Butt et al., 1992). This sometimes re-quires the application of extracellular concentrations be-tween 1 and 5 mM Bt2-cGMP or N2-Bt-cGMP (Francis etal., 1988). Under such conditions, activation of PKA cannotbe excluded (Kas of 50 mM and 25 mM for PKA-I and -II,respectively; Francis et al., 1988), and thus, can lead tomarked misinterpretations of experimental results if bothPKA and PKG are present.

Moreover, the butyrate itself, released from Bt2-cAMPand Bt2-cGMP, may produce several effects in cells that of-ten interfere with second messenger pathways. Butyrate wasreported to stimulate gene expression in several cell systems(Burns et al., 1988; Tamura & Cox, 1988; Feng et al.,1996), and to stimulate protein kinase C (Rivero & Adun-yah, 1998; Cuisset et al., 1998). Butyrate was also found toinduce differentiation and growth inhibition in cancer cells(Joshi et al., 1988; Hutt-Taylor et al., 1988; Kawamoto etal., 1998) and to initiate and potentiate apoptosis in humancarcinoma cells (Soldatenkov et al., 1998; Conway et al.,1998), effects that previously were correlated with tumorsensitivity toward Bt2-cAMP treatment (see Cho-Chung etal., 1995). Sodium butyrate, arginine butyrate, and the bu-tyrate prodrug tributyrin have been used in treatment of pa-tients with leukemia or solid tumors (see Conley et al.,

Table 1Lipophilicity data for cyclic nucleotide analogsa

Compound log KWb rLcAMP

c Compound log KW rLcGMPd

cAMP 1.09 1 cGMP 0.77 18-methylamino-cAMP 1.10 1 8-Br-cGMP 1.17 2.58-Cl-cAMP 1.32 1.7 PET-cGMP 2.47 508-Br-cAMP 1.35 1.8 8-pCPT-cGMP 2.52 56N6-Bt-cAMP 1.64 3.5 Bt2-cGMP 2.56 62N6-benzoyl-cAMP 1.90 6.5 8-Br-PET-cGMP 2.83 1158-piperidino-cAMP 2.17 12Bt2-cAMP 2.42 21 Sp-cGMPS 1.02 1.8N6-phenyl-cAMP 2.46 23 Sp-8-Cl-cGMPS 1.45 4.86-MBC-cAMP 2.60 32 Sp-8-pCPT-cGMPS 2.66 788-pCPT-cAMP 2.65 36 Sp-8-Br-PET-cGMPS 3.03 182Sp-cAMPS 1.32 1.7 Rp-cGMPS 0.89 1.3Sp-5,6-Cl2-cBIMPS 2.99 79 Rp-8-Cl-cGMPS 1.27 3.2

Rp-8-Br-cGMPS 1.29 3.3Rp-cAMPS 1.21 1.3 Rp-8-pCPT-cGMPS 2.60 68Rp-8-Cl-cAMPS 1.44 2.2 Rp-8-Br-PET-cGMPS 2.83 115Rp-8-Br-cAMPS 1.47 2.4Rp-8-pCPT-cAMPS 2.72 43

a Lipophilicity is expressed as log KW, where KW is the extrapolated capacity factor for 100% water in isocratic reversed-phase HPLC (for details, seeBraumann & Jastorff, 1985).

b log KW data taken from Genieser (1995), Krass et al. (1997), and Vaandrager et al. (1997).c Relative lipophilicity of cAMP analog compared with cAMP.d Relative lipophilicity of cGMP analog compared with cGMP.


1998). The use of butyryl-substituted cyclic nucleotides asbiochemical tools always requires control experimentswith butyrate. Compared with the dibutyryl compounds, N6-Bt-cAMP and N2-Bt-cGMP exhibit reduced stability tohydrolysis by PDEs and 59-nucleotidases, present in cells orin serum-supplemented cell culture medium. Thus, themonobutyrylated 59-monophosphates and nucleosidesshould be considered as relevant metabolites and, therefore,additionally tested in control experiments (Coulson et al.,1983; Zimmerman et al., 1985; Zorn et al., 1993).

In conclusion, both Bt2-cAMP and Bt2-cGMP are farfrom being ideal tools for cyclic nucleotide signal transduc-tion research due to low target specificity (Bt2-cGMP) andreleased bioactive metabolites, including butyrate (Bt2-cAMP and Bt2-cGMP).

3.1.2. 8-Bromo-cyclic AMP and 8-bromo-cyclic GMP8-Br-cAMP is known to selectively activate PKA-I and -II

in the mid-nanomolar range in vitro (Kas of 65 nM and 53 nM,respectively), while stimulation of PKGs requires concentra-tions z2 orders of magnitude higher (Kas of 5.8 mM, 10 mM,and 6 mM for PKG-Ia, -Ib, and -II, respectively; Corbin et al.,1986; Francis et al., 1988; Pöhler et al., 1995). 8-Br-cGMP ac-tivates the PKG isozymes with Ka values of 10–26 nM (PKG-Ia), 210–1000 nM (PKG-Ib), and 25 nM (PKG-II; Corbin etal., 1986; Francis et al., 1988; Sekhar et al., 1992; Butt et al.,1992; Pöhler et al., 1995). The wide range of activation con-stants, especially for PKG-Ib, is largely due to different de-

signs of kinase assays with respect to temperature, salt con-centration, substrates, and ATP concentration (Vaandrager etal., 1997). This is no specific 8-Br-cGMP effect, since the Kasfor cGMP itself demonstrate a similar pattern. Half-maximalstimulation of PKA-I and -II occurs at 2.8 mM and 12 mM8-Br-cGMP (Francis et al., 1988; Butt et al., 1992), leading tosufficient discrimination between PKA and PKG-Ia and -II(100-fold). However, with PKG-Ib and PKA-I, the selectivityof 8-Br-cGMP is significantly reduced, with only a 2.8- to 13-fold lower Ka for PKG-Ib. Furthermore, 8-Br-cGMP wasshown to activate the rod photoreceptor CNG channel conduc-tance efficiently (EC50 of 1.6 mM) when applied to the cytoso-lic side of excised patches of retinal rod outer segments (Zim-merman et al., 1985).

8-Br-cAMP exhibits only a 2-fold increased lipophilicitycompared with cAMP (Table 1), often leading to millimolarconcentrations for experiments with intact cells. 8-Br-cGMPis as polar as cAMP (Table 1), which implies an even lowercell permeability compared with 8-Br-cAMP. An importantobstacle is the fact that both 8-Br-cAMP and 8-Br-cGMPcan be hydrolyzed by certain PDEs (although relativelypoorly), generating the corresponding 59-monophosphateanalogs and, subsequently, the nucleoside analogs (Zimmer-man et al., 1985; Francis et al., 1988; Sandberg et al., 1991;Butt et al., 1992). Unfortunately, both compounds are stillfrequently described as highly membrane-permeable andstable against PDE hydrolysis, resulting in wrong interpre-tations of experimental data, especially with long-term incu-bations.

Taken together, the 8-bromo-substituted cyclic nucle-otide analogs are more advanced tools for signal transduc-tion research compared with the dibutyryl substituted com-pounds, due to reduced metabolic turnover by PDEs andexcellent activation potentials for PKA and PKG, combinedwith good (8-Br-cAMP) to moderate (8-Br-cGMP) enzymespecificity. However, insufficient membrane permeability isthe major limitation of these analogs for experiments withintact cell preparations.

3.2. Mapping studies as a key for improved cyclic nucleotide analogs

3.2.1. Cyclic AMP-dependent protein kinase Type I and Type IIThe majority of synthetic cyclic nucleotide analogs ex-

hibit low biological potency. Nevertheless, such analogswere successfully employed to define the molecular interac-tion potential between cAMP and cGMP and the cyclic nu-cleotide-binding domains of their target enzymes. Carefulcharacterization by means of cyclic nucleotides is consid-ered to be a prerequisite for the design of analogs able to ac-tivate or inhibit a given cyclic nucleotide-binding proteinwith high selectivity inside a cell (Schaap et al., 1993).Since PKA-I and -II are important modulators of prolifera-tion control in cancer cells and human cancers (Section 4.1),most mapping studies with cyclic nucleotides were per-formed with purified PKA holoenzymes and R-subunits.

Fig. 2. Chemical structures of widely used cyclic nucleotide analogs in signaltransduction research: Bt2-cAMP, Bt2-cGMP, 8-Br-cAMP, and 8-Br-cGMP.


The kinetics of each of the four individual cAMP-bindingsites (AI, AII, BI, BII) were studied with more than 100 cy-clic nucleotide analogs (De Wit et al., 1982, 1984; Yagura& Miller, 1981; Øgreid et al., 1985, 1989; Gjertsen et al.,1995). In general, analogs with hydrophobic substituents inposition N6 exhibited improved binding affinities for AI andAII and reduced affinities for BI and BII, compared withcAMP. Similar hydrophobic modifications at position C-8increased the affinity to BII, while some positively chargedsubstituents at C-8 produced high BI affinity. The situationis even more complex than summarized here (see Øgreid etal., 1989) and the subject of a current study (Schwede et al.,2000b).

Although no isozyme-specific activator for PKA-I or -II hasbeen described yet, each isozyme can be triggered by syner-gistic pairs of analogs, leading to preferential activation ofPKA-I or -II (Øgreid et al., 1985; Døskeland et al., 1991,1993). For example, the combination of 8-piperidino-cAMP(selective for AI and BII) with 8-methylamino-cAMP or8-aminohexylamino-cAMP (selective for BI) produces prefer-ential activation of PKA-I, while the combination of 8-piperi-dino-cAMP with N6-benzoyl-cAMP (selective for AI andAII) activates PKA-II. Two examples of PKA isozyme-directed synergistic pairs of analogs are shown in Fig. 3.This approach was successfully applied for identification ofthe specific PKA isoform involved in numerous biologicalprocesses (Lanotte et al., 1991; Vintermyr et al., 1995; Tor-gersen et al., 1997; Braun et al., 1998; Parvathenani et al.,1998; Singh et al., 1998; Steagall et al., 1998; Qi et al., 1999).

In bovine pinealocytes, the PKA-II-directed pair N6-phenyl-cAMP and Sp-5,6-dichloro-1-b-D-ribofuranosyl-benzimida-zole-39,59-cyclic monophosphorothioate (Sp-5,6-Cl2-cBIMPS)was reported to be as effective as the combination N6-phe-nyl-cAMP and 8-para-chlorophenylthio-cAMP (8-pCPT-cAMP) (Maronde et al., 1997). In contrast to N6-phenyl-cAMP and 8-pCPT-cAMP, the combination N6-phenyl-cAMPand Sp-5,6-Cl2-cBIMPS showed no signs of toxicity even athigher doses (Maronde et al., 1997). Very recently, an im-proved PKA-II-directed pair of analogs, N6-mono-tert-butyl-carbamoyl-cAMP (6-MBC-cAMP) combined with Sp-5,6-Cl2-cBIMPS, was successfully used in intact cells for the in-duction of phosphorylation of the transcription factor cAMPresponsive element-binding protein (CREB) and subsequentproduction of the hormone melatonin (Maronde et al.,1999), and also for the stimulation of the inhibitory tran-scription factor inducible cAMP early repressor (ICER)(Pfeffer et al., 2000) in pinealocytes. These findings are ofconsiderable interest since, in living cells, this combination(and the others mentioned before) behaved exactly as pro-posed from theoretical considerations and calculationsbased on binding affinities to PKA (Øgreid et al., 1985;Døskeland et al., 1991, 1993).

8-para-Chlorophenylthio-cAMP (8-pCPT-cAMP) re-ceived considerable importance as an excellent activator forPKA-I and -II, mainly due to its high lipophilicity and goodmembrane permeability (Table 1). Unfortunately, several

properties of this analog result in lacking PKA/PKG path-way specificity. 8-pCPT-cAMP is an almost equipotentstimulator of both PKA-II (Ka of 50 nM) and PKG-Ia (Ka of110 nM; Sandberg et al., 1991). Moreover, 8-pCPT-cAMPis a rather good inhibitor of PDEV (IC50 of 0.9 mM; Con-nolly et al., 1992), releases metabolites with unknown ef-fects (Coulson et al., 1983), and displays PDE stability com-parable with 8-Br-cAMP but is not, as often stated, fullystable against hydrolysis by PDEs (Francis et al., 1988;Sandberg et al., 1991). While the lack of PKA/PKG speci-ficity implies limited usefulness of this compound as a toolto distinguish between the PKA and PKG pathway, 8-pCPT-cAMP might be an interesting lead structure for some phar-macological applications in which parallel activation ofboth pathways is desired, e.g., organ preservation in trans-plantation (Section 4.3).

3.2.2. Cyclic GMP-dependent protein kinase Type Ia,Type Ib, and Type II

PKG-Ia was also tested with a multitude of cyclic nucle-otides (Corbin et al., 1986; Sekhar et al., 1992). Activationpotencies higher than cGMP itself (Ka of 110 nM) were pro-duced by analogs with hydrophobic substituents at positionC-8, like 8-Br-cGMP (Ka of 26 nM) and 8-para-chlorophe-nylthio-cGMP (8-pCPT-cGMP; Ka of 50 nM), or at posi-tions 1, N2 like the doubly modified b-phenyl-1,N2-etheno-cGMP (PET-cGMP; Ka of 26 nM). Interestingly, among all

Fig. 3. Synergistic pairs of cyclic nucleotide analogs for preferential activa-tion of (A) PKA-I: 8-methylamino-cAMP and 8-piperidino-cAMP and (B)PKA-II: 6-MBC-cAMP and Sp-5,6-Cl2-cBIMPS. Other examples of syner-gistic pairs of analogs are given in Section 3.2.1.


analogs tested, 8-Br-cGMP exhibited highest binding affinityto the slow cGMP-binding site (3.4-fold better than cGMP;slow site/fast site selectivity ratio, 8), while PET-cGMP wasthe compound with highest affinity to the fast cGMP-bind-ing site (10-fold better than cGMP; slow site/fast site selec-tivity-ratio, 1:6.3). However, not much emphasis has beendirected to studying potential synergism of these com-pounds towards PKGs. The terms fast and slow bindingsites are derived from dissociation studies with initiallybound cGMP. After dilution, one binding site rapidly ex-changes cGMP, while the other exhibits slower dissociationkinetics (Corbin et al., 1986). PKG-Ib and -II were mappedonly with a reduced number of cyclic nucleotides (Wolfe etal, 1989; Sekhar et al., 1992; Pöhler et al., 1995; Gamm etal., 1995; Vaandrager et al., 1997). Hence, knowledge aboutthese cGMP-binding sites is rather limited. For PKG-Ib,PET-cGMP, and some PET-cGMP analogs with additionalmodifications (e.g., 8-Br-PET-cGMP) exhibited highest ac-tivation potencies with Kas in the range of 9–20 nM (Sekharet al., 1992). PKG-II is best activated by 8-pCPT-cGMP(Kas of 3.5–80 nM, depending on ATP concentration and ki-nase preparation; Pöhler et al., 1995; Vaandrager et al.,1997). Similar to the situation in the PKA field, no isozyme-specific activator for PKGs is yet available. However, dif-ferent activation patterns for each kinase isozyme by a min-imum set of three or four cyclic nucleotides can be extractedout of several in vitro studies (Corbin et al., 1986; Francis etal., 1988; Sekhar et al., 1992; Gamm et al., 1995; Pöhler etal., 1995; Vaandrager et al., 1997) as follows:

PKG-Ia: 8-Br-cGMP/8-Br-PET-cGMP or PET-cGMP .8-pCPT-cGMP . (cGMP)

PKG-Ib: 8-Br-PET-cGMP or PET-cGMP .. (cGMP)/8-pCPT-cGMP/8-Br-cGMP

PKG-II: 8-pCPT-cGMP . 8-Br-cGMP . (cGMP) ..8-Br-PET-cGMP or PET-cGMP

Such different activation patterns by a small set of cGMPanalogs correlated well with their stimulation of PKG-II-medi-ated chloride secretion in intestinal epithelia (Vaandrager etal., 1997), their potency in relaxing pig coronary arteries,which is considered to be mediated by PKG-Ia (Sekhar etal., 1992; Francis & Corbin, 1994b), and might also be use-ful for PKG isozyme identification in other tissues or cells.

PET-cGMP, 8-Br-PET-cGMP, and especially 8-pCPT-cGMP (Fig. 4) demonstrate good resistance to hydrolysis bydifferent PDE isozymes (Francis et al., 1988; Butt et al.,1992, 1995b; Thomas et al., 1992), although not all PDEisozymes have been tested yet. PET-cGMP activates PKG-Ia and -Ib with Kas of 26 nM and 20 nM, respectively(Sekhar et al., 1992). Activation of PKA-I and -II requiresconcentrations z200-fold higher (Kas of 5.6 mM and 5 mMfor PKA-I and -II, respectively; Øgreid et al., 1985). How-ever, it does not discriminate between PKA isozymes andsome PKG-II preparations (Ka PKG-II, 4.2–4.7 mM; Vaan-drager et al., 1997; Gamm et al., 1995). 8-Br-PET-cGMP ex-hibits best activation constants for PKG-Ia and -Ib (Kas of

13 nM and 9 nM, respectively) amongst all commerciallyavailable cGMP analogs (Sekhar et al., 1992). Like PET-cGMP, 8-Br-PET-cGMP activation of PKG-II requireshigher concentrations (Ka of 1.6 mM), leading to significantPKG-I selectivity versus PKG-II for both compounds. Since8-Br-PET-cGMP is 2-fold more lipophilic (Table 1), comparedwith PET-cGMP, it appears likely that this compound is thebest choice to trigger PKG-I activation in vivo. 8-pCPT-cGMP activates PKG-Ia and -II with Kas of 40–50 nM and3.5–80 nM, exhibiting a specificity factor of 10–2000 com-pared with PKA-I and -II (Kas of 0.74 mM and 1.3–7 mM, re-spectively; Francis et al., 1988; Butt et al., 1992; Sekhar etal., 1992; Pöhler et al., 1995; Vaandrager et al., 1997). Nospecificity is found for PKG-Ib (Ka of 440–900 nM) andPKA isozymes.

In conclusion, no single compound alone, but combina-tions of 8-Br-PET-cGMP or PET-cGMP with 8-Br-cGMPand 8-pCPT-cGMP, are suggested as powerful tools tostudy PKG action and to identify PKG isozymes involved inbiological processes.

3.2.3. Mapping studies with testkit analogs and thediscovery of Rp-adenosine 39,59-cyclicmonophosphorothioate

The selection of cyclic nucleotides tested as regulators ofPKAs, PKGs, some PDEs (Francis et al., 1990; Thomas etal., 1992), and CNG ion channels (Kaupp & Koch, 1986;Caretta et al., 1985; Tanaka et al., 1989; Brown et al., 1993)was largely based on availability, and was more random thansystematic. A different approach, the so-called testkit con-cept, used only a limited set of cyclic nucleotide analogswith systematic variations of molecular interaction potentialsdonated by cAMP or cGMP. The main idea of this concept isto selectively manipulate the hydrogen bonding characteris-tics of each relevant atomic group in cAMP and cGMP, andin addition, their charge-transfer interaction potential, hydro-phobicity, bulkiness, and dipole moment using ,20 cyclicnucleotide analogs (see Jastorff et al., 1992). A multitude of

Fig. 4. Chemical structures of PKG activators: PET-cGMP, 8-Br-PET-cGMP, and 8-pCPT-cGMP.


cAMP- and cGMP-binding proteins have been studied bythese testkit compounds (Jastorff et al., 1979; De Wit et al.,1982, 1984; Erneux et al., 1984; Scholübbers et al., 1984;Van Lookeren Campagne et al., 1990; Van Ments-Cohen etal., 1991; Nass et al., 1992; Butt et al., 1995a; Beltman et al.,1995; Feldwisch et al., 1995; Hebert et al., 1998), leading toidentification of several different types of cAMP- andcGMP-binding sites. For instance, testkit analogs were suc-cessfully employed for systematic mapping of PKA-I, lead-ing to identification of the essential molecular interactions(pharmacophore) between cAMP and the cyclic nucleotide-binding sites AI and BI (Jastorff et al., 1979; De Wit et al.,1982). The proposed model for the pharmacophore was thebasis for the design of several second-generation cAMP ana-logs with improved selectivities (Sections 3.2.1 and 3.3).Furthermore, X-ray analysis of a deletion mutant of the RIa-subunit bound to cAMP confirmed most of the essential in-teractions proposed in 1979 (Su et al., 1995).

Using this concept, the first antagonist of PKA, Rp-cAMPS,with a sulfur atom replacing oxygen at the equatorial exocy-clic position of the cyclic phosphate, was identified. In con-trast, the corresponding Sp-isomer (Sp-cAMPS), with sulfurreplacing oxygen at the axial exocyclic position (see Fig. 5),turned out to be an activator of both PKAs (De Wit et al.,1982; O’Brian et al., 1982).

In summary, mapping and testkit studies provided a signifi-cant increase of knowledge about the cyclic nucleotide-bindingmotifs of several cNMP target proteins and promoted thedevelopment of improved cAMP and cGMP analogs as acti-vators for PKA and PKG. The identification of the first antag-onist of PKA, and the subsequent development of a newgeneration of Rp/Sp-cAMPS and Rp/Sp-cGMPS analogs,was then initiated (Sections 3.3 and 3.4). However, someCNG channels, and especially the recently identified PDEfamilies 8-10, HCN channels, as well as Epac/cAMP-GEFproteins, have not yet been systematically characterizedwith cyclic nucleotides.

3.3. Rp- and Sp-adenosine 39,59-cyclic monophosphorothioate analogs as new biochemical tools

Rp-cAMPS and Sp-cAMPS were synthesized amidst thefirst wave of cyclic nucleotide analog design in the 1970s

(Eckstein et al., 1974). This study was the first to show thatphosphorothioate-modified cyclic nucleotides are muchmore resistant toward mammalian cyclic nucleotide PDEs.The two isomers behaved differently in that the Rp-diastere-omer inhibited PKA, whereas the Sp-diastereomer was aweak agonist (De Wit et al., 1982; O’Brian et al., 1982). Thisled shortly to the finding that Rp-cAMPS efficiently inhib-ited glucagon- (or cAMP-) induced glucose production (withan apparent Ki of 8.3 mM; Rothermel et al., 1983, 1984; seeBotelho, 1986). The current mechanistic model for Rp-cAMPS action suggests competitive binding to the cAMP-binding sites of the PKA holoenzyme without efficiently in-ducing the conformational event leading to the release andactivation of the C-subunits (De Wit et al., 1982; Rothermel& Botelho, 1988; Dostmann et al., 1990; Dostmann & Tay-lor, 1991; Neitzel et al., 1991; Dostmann, 1995). However,broader use of this new tool was hampered by its poor mem-brane permeability (comparable with 8-Br-cAMP; Table 1).New synthetic methods developed for Rp-cAMPS and itsderivatives (Genieser et al., 1988, 1989), and their commer-cial availability, made these PKA inhibitors widely usedtools for investigation of cAMP-mediated effects.

A comparative study of some of these new Rp-cAMPSderivatives in fibroblasts, promyelocytic leukemia cells, andhepatocytes, using different assay systems, exemplifiessome fields where different Rp-cAMPS derivatives may beuseful (Gjertsen et al., 1995). In particular, Rp-8-Br-cAMPS and Rp-8-Cl-cAMPS were superior to Rp-cAMPSas antagonists of purified PKA-I and cell lines with a highPKA-I/PKA-II ratio, and, therefore, were proposed to re-place Rp-cAMPS as first-line antagonists of PKA-I.

Several hundred studies successfully employed Rp-cAMPS in vitro and in vivo (e.g., Botelho et al., 1988;Schaap et al., 1993; Bito et al., 1996; Kaji et al., 1997; Ming etal., 1997; Vischer & Wollheim, 1998; Boulanger & Poo, 1999).A more complete reference list can be found on the WorldWide Web at the following URL: http://www.biolog.de/a002ref.html.

In some research fields, Rp-cAMPS and its derivativeshave brought considerable progress to understanding theunderlying molecular mechanisms. Here we briefly reviewsome of these aspects.

The first area is concerned with experimentally inducedaddiction behaviour. The basic mechanisms involved, theimportant distinction between short- and long-term effects,and the site of action in the brain have been reviewed else-where (Nestler, 1992; Nestler & Aghajanian, 1997). Inbrief, infusions of Rp-cAMPS into the periaqueductal graymatter significantly attenuated prominent behavioural signsof morphine withdrawal, whereas Sp-cAMPS induced a“quasi-withdrawal syndrome” (Punch et al., 1997). More-over, bilateral infusions of Rp-cAMPS into the nucleus ac-cumbens reduced, whereas Sp-cAMPS increased, baselinecocaine self-administration (Self et al., 1998).

Another interesting paradigm is the so-called long-termpotentiation. The hippocampal long-term potentiation is aFig. 5. Chemical structures of Rp-cAMPS and Sp-cAMPS.


process thought to be related to some forms of learning andmemory formation. Among other PKA inhibitors, Rp-cAMPS was shown to inhibit this process, while Sp-cAMPSpromoted it (Matthies & Reymann, 1993; Weisskopf et al.,1994; Blitzer et al., 1995; Bito et al., 1996; Nguyen & Kan-del, 1997; Huang & Kandel, 1998; Ma et al., 1999).

Rp-cAMPS is also a valuable tool in diabetes research,especially on glucagon-like peptide (GLP)-1 as a novel thera-peutic treatment of diabetes mellitus Type 2. It has been shownthat GLP-1-induced insulin release from pancreatic islets isinhibited by Rp-8-Br-cAMPS (Fig. 6; Ding & Gromada, 1997;Ding, W.-G. et al., 1997; Holz et al., 1999). The assumptionthat the GLP-1 effect is mediated at least in part by cAMP issupported by the finding that Sp-cAMPS (Sjöholm, 1992,1997) and Sp-5,6-Cl2-cBIMPS (Fig. 3) induced insulin releasefrom these preparations (Laychock, 1993).

Finally, one example of many cAMP-mediated hormonalprocesses inhibitable by Rp-cAMPS is pineal melatonin pro-duction. Nocturnal adrenergically induced production of me-latonin is mediated by cAMP, and this induction is inhibitedby Rp-cAMPS and its analogs (Roseboom & Klein, 1995;Maronde et al., 1997, 1999; Wicht et al., 1999). Moreover,both phosphorylation of the activating transcription factorCREB, leading to up-regulation of the expression of N-ace-

tyltransferase, the rate-limiting enzyme for melatonin produc-tion, and induction of the later-appearing inhibitory transcrip-tion factor ICER (see Stehle et al., 1993; Pfeffer et al., 2000)are mediated by PKA-II. This has been demonstrated by bothsite-selective activators of PKA-I and -II (Maronde et al.,1997, 1999; Pfeffer et al., 2000) and by the use of Rp-8-pCPT-cAMPS (Fig. 6), which is a better inhibitor of PKA-IIthan -I (Gjertsen et al., 1995; Maronde et al., 1997, 1999).

Taken together, among the different commercially avail-able derivatives of Rp-cAMPS, Rp-8-Cl-, Rp-8-Br-, andRp-8-pCPT-cAMPS have been widely used and merit men-tion as derivatives of choice for in vitro and in vivo PKA in-hibition. Other types of PKA inhibitors, the ATP-site com-petitors H-8, H-89, and KT 5720, are quite common as well.H-89 especially is widely used for inhibition of PKA, but itis not as specific for PKA as Rp-cAMPS analogs, and manyside effects have been reported (Hemmings, 1996; Boundyet al., 1998; Hussain et al., 1999; Bode et al., 1999).

Among the Sp-cAMPS derivatives, Sp-cAMPS, the par-ent compound, is still most widely used as a cAMP agonist.Nevertheless, we feel that the best specific activator for PKAis Sp-5,6-Cl2-cBIMPS (Section 3.2.1). It is highly lipophilic(Table 1) to ensure membrane passage and most resistant tohydrolysis by PDEs, activates PKA at least as well as cAMP,and is a 300-fold less potent activator of PKG (Sandberg etal., 1991; Genieser et al., 1992). Moreover, Sp-5,6-Cl2-cBIMPS (Fig. 3) has been shown to be washed out easily, aproperty very valuable in kinetic studies (Schultz et al., 1994).

3.4. Rp- and Sp-guanosine 39,59-cyclic monophosphorothioate analogs as new biochemical tools

The identification of Rp-cAMPS as an inhibitor of PKA notonly initiated the search for better Rp-cAMPS analogs withhigher specificities, but also the synthesis of Rp-cGMPS andRp-8-Cl-cGMPS (Genieser et al., 1988). Both compoundswere identified as competitive inhibitors of PKG-Ia (Butt etal., 1990). In analogy with PKA, these analogs are assumed tobind to PKG, but apparently do not evoke the conformationalchanges of the enzyme required for activation of the C-subunit(Zhao et al., 1997). Ki values of Rp-cGMPS analogs aresummarized in Table 2. For better comparison, only data forPKG-Ia (bovine lung) and PKA-II (bovine heart) from onelaboratory are included. Rp-cGMPS is only a weak and non-specific antagonist, since it inhibits both PKG-Ia and PKA-II,with Kis of 20 mM. Rp-8-Cl-cGMPS has improved properties,with a Ki of 1.5 mM for PKG-Ia and 66-fold selectivity forPKG versus PKA-II (Ki of 100 mM for PKA-II; Butt et al.,1990), comparable with Rp-8-Br-cGMPS, a PKG antagonistthat is more frequently used in signal transduction research(Kis of 3.7 mM [PKG-Ia], and 25 mM [PKA-II]; Kawada et al.,1997). However, poor membrane permeability remains a majorlimitation of both halogenated compounds for experimentswith living cells. Recently, this problem was tackled with thedevelopment of two lipophilic PKG antagonists; namely,Rp-8-pCPT-cGMPS (Butt et al., 1994) and Rp-8-Br-PET-

Fig. 6. Chemical structures of Rp-cNMPS analogs proposed as first-line inhib-itors with improved isozyme selectivities for PKA or PKG: Rp-8-Br-cAMPS(PKA-I; Gjertsen et al., 1995), Rp-8-pCPT-cAMPS (PKA-II; Gjertsen et al.,1995; Maronde et al., 1997, 1999), Rp-8-Br-PET-cGMPS (PKG-Ia and -Ib;Butt et al., 1995b; Vaandrager et al., 1997), and Rp-8-pCPT-cGMPS (PKG-II;Gamm et al., 1995; Vaandrager et al., 1997).


cGMPS (Butt et al., 1995b; see Fig. 6). These analogs exhibit68-fold (Rp-8-pCPT-cGMPS) and 115-fold (Rp-8-Br-PET-cGMPS) increased lipophilicity compared with cGMP (Ta-ble 2). Rp-8-pCPT-cGMPS demonstrated similar inhibitionconstants for all PKG isozymes (Kis of 0.5 mM, 0.6 mM, and0.29–0.5 mM for PKG-Ia, -Ib, and -II, respectively; Butt et al.,1994; Pöhler et al., 1995; Vaandrager et al., 1997), comparedwith a Ki of 8.3 mM for PKA-II (15- to 30-fold selectivity forPKGs). However, completely different results of isozyme-selective inhibition of PKG-II (Ki of 0.16 mM) comparedwith PKG-Ia (Ki of 18.3 mM) were reported by Gamm et al.(1995). The latter study indicated the potential usefulness ofRp-8-pCPT-cGMPS for characterization of PKG isozymes.Among all phosphorothioate cGMP analogs tested so far,Rp-8-Br-PET-cGMPS was found to be the most potent in-hibitor for PKG-Ia and -Ib (Kis of 0.035 mM and 0.03 mM,respectively), combined with a more than 300-fold discrimina-tion of PKA-II (Ki of 11 mM; Butt et al., 1995b), while inhibi-tion of PKG-II occurred at a significantly higher concentration(Ki 0.9 mM; Vaandrager et al., 1997).

Taken together, Rp-8-pCPT-cGMPS and Rp-8-Br-PET-cG-MPS can be considered as potent PKG inhibitors, especiallyuseful for experiments with intact cells. Both selectively in-hibited PKG in intact human platelets (Butt et al., 1994) andin intestinal mucosa (Vaandrager et al., 1997), two systemsthat are known to contain high levels of PKG and PKA(Eigenthaler et al., 1992; Forte et al., 1992). In rat tail arteriesand rabbit portal vein, Rp-8-Br-PET-cGMPS selectively inhib-ited PKG, while PKA-mediated effects were not modulated atsimilar concentrations (Butt et al., 1995b; Ruiz-Velasco et al.,1998). The combined use of both of these Rp-cGMPS analogsmay be seen as a novel approach to distinguish betweenPKG-I- and -II-action in cells and tissues. However, it shouldbe recognized that high extracellular concentrations of Rp-8-pCPT-cGMPS (0.5–1 mM) and Rp-8-Br-PET-cGMPS(.0.2 mM) can also at least partially inhibit PKA signaling(Smolenski et al., 1998). Selective inhibition of PKG needs tobe carefully established in each system by control experiments,including biochemical methods, suitable PKA antagonists,and PKG/PKA agonists (see Smolenski et al., 1998).

Among the Sp-cGMPS analogs (Table 3), Sp-8-pCPT-cGMPS and Sp-8-Br-PET-cGMPS are considered as first-linecompounds, due to PDE resistance, lipophilicity, and activa-tion constants for PKGs. Only Sp-8-Br-PET-cGMPS displayssufficient selectivity for PKG versus PKA (Kas: PKG-Ia,2.6 mM; PKG-Ib, 2.5 mM; PKA-II: .1000 mM; Butt et al.,

1995b), and, in addition, inhibits the rod photoreceptorcGMP-gated channel (IC50, 105 mM; Wei et al., 1996). This islargely due to the PET substituent in positions 1,N2 of theguanine base, which counteracts the well-established positiveeffect of substituents at position 8 for channel activation(Zimmerman et al., 1985; Tanaka et al., 1989; Wei et al., 1998).Therefore, this compound could also be useful for discrimi-nation between PKG and cGMP-gated channels. In contrast,Sp-8-pCPT-cGMPS is not selective for PKG activation, andthus, should be used only if no PKG/PKA specificity, butsuperior PDE resistance, is required (Kas: PKG-Ia, 9.1-18mM; PKG-Ib, 1.8 mM; PKA-II, 4.6 mM; Butt et al., 1994;Gamm et al., 1995).

Selective activation of cNMP targets can also be achievedby the so-called polymer-linked dimer strategy (Kramer &Karpen, 1998). In this novel approach, two cGMP moleculesare linked by polyethylene glycol spacers of differentlengths. The authors showed that each cGMP target proteininvestigated (olfactory and rod CNG channels and PKG-Ia)requires a certain critical spacer length to exert full activa-tion. cGMP polymer-linked dimers of optimal length can be1000-fold better activators of the target than cGMP itself(Kramer & Karpen, 1998). This approach seems very prom-ising for the development of even more selective activators,and perhaps also inhibitors of cNMP target proteins. How-ever, the in vivo usefulness of these compounds currently isunknown, since membrane permeability and stability againstenzymatic degradation have not been investigated.

3.5. Bioactivatable, membrane-permeant prodrugsof cyclic nucleotides

Two different synthetic approaches frequently are usedto increase the membrane permeability of cyclic nucle-otides. The dominant strategy was to introduce hydrophobicsubstituents attached mainly at the adenine or guanine moi-ety of cAMP and cGMP (e.g., the 8-para-chlorophenylthio-group in cAMP and cGMP, and the b-phenyl-1,N2-etheno-group in cGMP). Some of these analogs combine increasedmembrane permeability with improved binding affinities totheir receptor proteins and decreased susceptibility to hy-drolysis by PDEs (Sections 3.2.1 and 3.2.2).

Another approach is to modify the negatively chargedphosphate diester, the group mainly responsible for the im-permeability of cAMP and cGMP. Esterification of the cyclicphosphate of cAMP has been reported (Gohil et al., 1974;

Table 2Kis (in mM) of Rp-cGMPS and Rp-cGMPS analogs for PKG-Ia and PKA-II

Compound PKG-Ia PKA-II

Rp-cGMPS 20 20Rp-8-Cl-cGMPS 1.5 100Rp-8-pCPT-cGMPS 0.5 8.3Rp-8-Br-PET-cGMPS 0.035 11

Data from Butt et al. (1990, 1994, 1995b).

Table 3Ka (in mM) of Sp-cGMPS and Sp-cGMPS analogs for PKG-Ia and PKA-II

Compound PKG-Ia PKA-II

Sp-cGMPS 27 27Sp-8-Cl-cGMPS 3.4 8.3Sp-8-pCPT-cGMPS 18 4.6Sp-8-Br-PET-cGMPS 2.6 .1000

Data from Butt et al. (1990, 1994, 1995b).


Gillen & Nagyvary, 1976), including alkylation to benzyl tri-esters of cAMP (Engels & Schläger, 1977), which has beensuccessfully tested on guinea-pig papillary muscle (Korth &Engels, 1987). Formation of the cyclic nucleotide in this caseappears to be a result of the chemical lability of the phosphatetriester. Considerable work has been done on photolyzableesters (so-called caged cyclic nucleotides), including o-nitro-benzyl esters (Engels & Schläger, 1977; Nerbonne et al.,1984), 1-(o-nitrophenyl)-ethylidene esters, and 4,5-dimethoxy-2-nitrobenzyl esters (Woottoon & Trentham, 1989; Hagen etal., 1996). After cell penetration, the charged cyclic nucleotideis liberated by UV light (Kao & Adams, 1993). If this transitionto the cyclic nucleotide is kinetically fast and complete, cagedcAMP and cGMP, and some analogs, provide excellent tools,especially for studies of CNG ion channels (Kaupp et al., 1998).

Recently, the bioactivatable acetoxymethyl (AM)-estermasking group, originally developed for the modification ofcarboxylic acids such as penicillin (Jansen & Russell, 1965)and polycarboxylic acids such as Fura-2 (Tsien, 1981, 1993),has been successfully introduced into Bt2-cAMP, Bt2-cGMP(Schultz et al., 1993), cAMP (Schultz et al., 1994), and cGMP(Zhuo et al., 1994). This synthetic approach of Schultz and co-workers was further extended to 8-substituted derivatives ofcAMP (Kruppa et al., 1997), derivatives of cGMP (Schwedeet al., 2000), and Sp-cAMPS (E. Maronde, P. Niemann, H.-W.Korf, & H.-G. Genieser, submitted for publication).

Alkylation and masking of the negative charge of the cy-clic phosphate with the AM-ester group leads to significantlyincreased lipophilicity and bioavailability (Kruppa et al.,1997). Inside the cell, the bioactivatable AM-esters are hy-drolyzed by endogenous esterases, with formation of aceticacid, formaldehyde and the parental cyclic nucleotide(Schultz et al., 1994). Given the fact that the liberated cyclicnucleotide is hardly able to leave the cell due to its polarity, acertain accumulation was anticipated and proven by HPLCanalysis of cell lysates of C6-glioma cells (M. Bartsch, un-published results). In this study, a 3.6-fold higher intracellularconcentration (110 mM) of the biologically active N6-Bt-cAMP was detected after a 1-hr incubation with extracellu-larly applied 30 mM Bt2-cAMP AM-ester (Bt2-cAMP/AM).

The bioactivatable membrane-permeant AM-ester pro-drug Bt2-cAMP/AM (Fig. 7) was 2–3 orders of magnitudemore potent than Bt2-cAMP in 3 model systems for cAMPaction (Schultz et al., 1993). Approximately 50% dissociation(activation) of fluorescent-labeled PKA, microinjected intofibroblasts, was achieved with 1 mM Bt2-cAMP/AM com-pared with 1000 mM Bt2-cAMP. Activation of intestinal Cl2

transport in human colon epithelial T84-cells was achievedwith EC50 values of 2 mM and 400 mM for Bt2-cAMP/AMand Bt2-cAMP, respectively. Half-maximal dispersion of pig-ment granules in angel fish melanophores was induced with10 mM Bt2-cAMP/AM compared with no effect with Bt2-cAMP, even at 1000 mM (Schultz et al., 1993, 1994). 8-pCPT-cAMP/AM, representing a combination of the good activatorof PKA, 8-pCPT-cAMP, with the improved bioavailabilityof the AM group, is currently the most potent cyclic nucleotide

prodrug to activate Cl2 transport in T84-cells, with an EC50 of150 nM (Kruppa et al., 1997). A concentration of 1 mMcGMP/AM (Fig. 7) was reported to be as effective as 250 mMBt2-cGMP or 100 mM 8-Br-cGMP for induction of long-termpotentiation in rat hippocampal neurons of the CA 1 region(Zhuo et al., 1994). Furthermore, cGMP/AM inhibited plateletaggregation with an EC50 of 1 mM compared with 500–1000mM for 8-Br-cGMP (C. Schultz & R. Y. Tsien, unpublishedresults). Sp-cAMPS/AM exhibited z6-fold better induction ofCREB phosphorylation in rat pinealocytes than the highlylipophilic Sp-5,6-Cl2-cBIMPS (E. Maronde, P. Niemann,H.-W. Korf, & H.-G. Genieser, submitted for publication),indicating that the negative charge at the cyclic phospho-rothioate of the latter compound still hampered membranepermeability.

Due to the fast metabolic turnover of cAMP and cGMP inintact cells, cAMP/AM and cGMP/AM should be especiallyuseful to study cellular processes where a transient intracel-lular cAMP or cGMP signal is required, while AM-esters ofcyclic nucleotides with increased metabolic stability (Sp-cAMPS/AM, 8-pCPT-cAMP/AM) are more useful in long-term applications, such as inhibition of cell growth or activa-tion of transcription factors.

Taken together, cyclic nucleotide esterification with theAM-ester group is the first step to significantly improve thetransmembrane delivery of cyclic nucleotides into intactcells. It can be speculated that the combination of cAMP/cGMP analogs with high in vitro activities for, e.g., PKA/PKG with the AM-ester masking group, or other even morelipophilic bioactivatable masking groups (Srivastva & Far-quhar, 1984; Thomson et al., 1993), might further reducethe required extracellular doses. Therefore, this latest gener-ation of cyclic nucleotide analogs may gain importance aspharmacological tools and therapeutics in the future.

4. Cyclic nucleotide analogs as prospective drugs

4.1. 8-Chloro-cyclic AMP, a new anticancer drug?

In the late 1980s, Cho-Chung and colleagues discoveredthat 8-Cl-cAMP, a potent analog of cAMP, initiated growth

Fig. 7. Chemical structures of the membrane-permeant, bioactivatablecyclic nucleotide derivatives Bt2-cAMP/AM and cGMP/AM.


inhibition in vitro and in vivo in a broad spectrum of humancarcinomas (breast, colon, lung), fibrosarcomas, and leukemiasat micromolar concentrations (Katsaros et al., 1987; Ally et al.,1988, 1989; Tagliaferri et al., 1988; Tortora et al., 1988; Cho-Chung et al., 1989). Other cAMP analogs, such as 8-pCPT-cAMP, 8-thiomethyl-cAMP, 8-thioisopropyl-cAMP, N6-benzyl-cAMP, N6-carbamoylphenyl-cAMP, or the doublymodified analog N6-phenyl-8-pCPT-cAMP, exhibited similarpotencies in the micromolar range, but further studies wereconducted mainly with 8-Cl-cAMP, most readily available bysynthesis. These results strongly reinforced interest in cyclicnucleotides as therapeutics against cancer. The results with8-Cl-cAMP encouraged researchers to embrace a new ap-proach to therapy in the treatment of cancer by nontoxicagents, which are able to restore the natural balance of pro-liferation and differentiation in cancer cells.

The importance of programmed cell death (apoptosis) asone of the main molecular mechanisms of action of chemo-therapeutics in the treatment against cancer was not yet rec-ognized in 1989 (Lowe et al., 1993). Subsequently, severalstudies reported apoptotic cell death related to 8-Cl-cAMPapplication to cancer cells (Lanotte et al., 1991; Bøe et al.,1995; Vintermyr et al., 1995; Ruchaud et al., 1995; Han etal., 1996; Hoffmann et al., 1996; Krett et al., 1997; Tortoraet al., 1997).

8-Cl-cAMP can discriminate between the two cAMP-binding sites (sites A and B) on the R-subunits (RI and RII)of PKA-I and -II (Ally et al., 1988). 8-Cl-cAMP binds withsimilar high affinity to both sites A and B of RI. In contrast,it binds with high affinity to site B of RII, but with low af-finity to site A, which may keep this isozyme in its nonacti-vated holoenzyme form. Ally et al. (1988) reported Kas of39.6 nM for PKA-I from rabbit skeletal muscle comparedwith 128.5 nM for PKA-II from bovine heart, indicating a3.3-fold selectivity for PKA-I. However, Døskeland and co-workers reported similar activation constants of 8-Cl-cAMPfor both PKAs prepared from essentially the same tissues,with Kas of 39.6 nM and 36 nM for PKA-I and -II, respec-tively (Øgreid et al., 1985). Overexpression of PKA-I hasbeen observed in a spectrum of human cancer cell lines, andhas been correlated with cell transformation and prolifera-tion (Gharrett et al., 1976; Russell, 1978; Ramage et al.,1995), while growth arrest and differentiation were linkedto elevated PKA-II concentrations or an increased ratio ofRII/RI (Lee et al., 1976; Cho-Chung, 1980, 1990). Overex-pression of the RIa-subunit is an accepted marker for poorprognosis in breast cancer patients (Miller et al., 1993). 8-Cl-cAMP was found to modulate RI and RII levels, leading torestoration of a more natural RII/RI balance in cancer cells.8-Cl-cAMP is able to down-regulate RIa, perhaps by facili-tating the degradation of the protein after its dissociationfrom the PKA holoenzyme, while RIIb expression is up-regulated at the transcriptional level (Cho-Chung et al.,1989; Rohlff et al., 1993; Ciardiello & Tortora, 1998) or notaffected (Noguchi et al., 1998), both leading to an increasedRII/RI intracellular ratio. The increased concentration of

RIIb in the cytosol of some human cancer cell lines is fol-lowed by rapid translocation of RIIb into the nucleus (Allyet al., 1988; Yokozaki et al., 1989), where RIIb is thought toplay a role in the restoration of normal gene transcription(Cho-Chung & Clair, 1993; Cho-Chung et al., 1995; Srivas-tava et al., 1998a). This translocation has not been con-firmed by any other group, and is currently believed to be anartefact (Hagiwara et al., 1993; Harootunian et al., 1993).

Nevertheless, growth arrest and differentiation by 8-Cl-cAMP is accompanied by inhibition of the expression of on-cogenes and growth factors, including ras, myc, erbB2,transforming growth factor-a, vascular endothelial growthfactor, and basic fibroblast growth factor (Tagliaferri et al.,1988; Ally et al., 1989; Tortora et al., 1989; Ciardiello et al.,1990, 1993; Rohlff et al., 1993; Bianco et al., 1997) in atime- and dose-dependent manner at the mRNA level (Ciar-diello et al., 1990; Bianco et al., 1997). In different leuke-mia cell lines (HL-60, MOLT-4, K-562, myc-K562), growthinhibition by 5–20 mM 8-Cl-cAMP was reported not to beassociated with cell killing, but with growth arrest. HL-60cells reacted with increased expression of differentiationmarkers, and a down-regulation of the RI subunit of PKAwas verified in all cell lines, while the RII levels were notaltered (Tortora et al., 1988).

The promising results of Cho-Chung and co-workers led tothe choice of 8-Cl-cAMP as the first cyclic nucleotide-basedpreclinical antineoplastic agent by the National CancerInstitute (Bethesda, MD, USA) in 1988, 3 decades after thesynthesis of the first analog of cAMP (Malspeis & Kemmenoe,1990; Tomaszewski et al., 1991; Cho-Chung, 1992; Cum-mings et al., 1994). In the period 1995–1997, the results ofseveral Phase I clinical trials with 8-Cl-cAMP were reported(Saunders et al., 1995, 1997; Tortora et al., 1995; Cummingset al., 1996), and currently, this cAMP analog is being testedin Phase II clinical trials (McDaid & Johnston, 1999).

However, there is still a long-standing controversy con-cerning the above proposal for the molecular basis of actionof 8-Cl-cAMP treatment. Several reports demonstrated that8-Cl-adenosine largely accounted for the growth inhibitory ef-fect of 8-Cl-cAMP in CHO cells, Molt-4 lymphoblasts (VanLookeren Campagne et al., 1991), human and rat gliomacells (Langeveld et al., 1992), colon cancer cells (Taylor &Yeoman, 1992), mouse lung epithelial cells (Lange-Carter etal., 1993), MCF-7 cells (Bøe et al., 1995; Vintermyr et al.,1995), and human promyelocytic leukemia NB4 cells (Hoff-mann et al., 1996). The metabolic pathway of 8-Cl-cAMP to8-Cl-adenosine in cell culture is controlled by 39,59-cyclicphosphodiesterase and 59-nucleotidase isozymes present inserum-supplemented medium or inside cultured cells. InMCF-7 cell culture experiments, an initial incubation with100 mM 8-Cl-cAMP led to the formation of 17 mM 8-Cl-ade-nosine after 72 hr in 5% fresh fetal calf serum, comparedwith 4.5 mM in heat-inactivated serum (Vintermyr et al.,1995). The use of heat-inactivated serum or addition of ade-nosine deaminase, an enzyme that converts 8-Cl-adenosineto inactive 8-Cl-inosine, was often linked with reduced po-


tency in growth inhibition by 8-Cl-cAMP (Bennett et al.,1985; Langeveld et al., 1992, 1997; Vintermyr et al., 1995;Juranic et al., 1998). The mode of action of 8-Cl-adenosineis also not well understood. However, it is reported to de-crease the RI-subunit of PKA in lung epithelial cells andthus, to modulate the RII/RI ratio (Lange-Carter et al.,1993), an effect previously attributed only to 8-Cl-cAMP.On the other hand, growth inhibition by 8-Cl-cAMP of c-rastransformed fibroblasts (MP3/3T3) was accompanied by re-duction of RI and cell cycle arrest in the G0/G1 phase, tworegulatory effects that were not mimicked by 8-Cl-adenosine(Noguchi et al., 1998). In this study, 8-Cl-cAMP producedgrowth inhibition only in transformed fibroblasts, while 8-Cl-adenosine was equally cytotoxic to both transformed andnontransformed cells, an effect also reported by others (e.g.,Han et al., 1993).

In summary, it is still not resolved whether 8-Cl-cAMPacts as a pharmacon itself or, at least in part, as a prodrug for8-Cl-adenosine. In addition, several of the above-mentionedstudies indicate cell-type specific differences of both com-pounds.

Two Phase I clinical trials, on patients with different solidtumors refractory to conventional therapy, were designed toevaluate the maximum-tolerated dose, pharmacokinetics, tox-icity, and potential biomodulatory effects of 8-Cl-cAMP(Tortora et al., 1995; Saunders et al., 1995). The maximum-tolerated doses in schedules with continuous infusions werefound to be between 0.045 mg/kg/hr (Saunders et al., 1995)and 0.2 mg/kg/hr (Tortora et al., 1995). Reversible hypercal-caemia in several patients and renal dysfunction (proteinuria)in some cases turned out to be the dose-limiting toxic effects.The hypercalcaemia probably is related to the parathyroidhormone, which was down-regulated in some patients, indi-cating possible interference with hormonal regulation. Thisphenomenon recently was studied in detail in a Phase I trialby Saunders et al. (1997). 8-Cl-cAMP was given to 16 pa-tients, and indeed, it was demonstrated that it exhibited a para-thyroid hormone-like effect of increased biosynthesis of 1,25-dihydroxyvitamin D, leading to hypercalcaemia, which wasreversible by discontinuation of the drug. Tortora et al. (1995)reported 8-Cl-cAMP plasma concentrations in the range of0.65–3 mM, depending on the protocol of administration.These concentrations were not toxic (with the exception ofhypercalcaemia) and were active in in vitro studies againsthuman cancer cell lines (Cho-Chung et al., 1989; Yokozaki etal., 1993; Hoffmann et al., 1996). Unfortunately, the designof this study did not consider potential formation of 8-Cl-ade-nosine in the plasma or tumors. Tortora et al. (1995) reportedantitumor activity in 4 (3 colon cancers, 1 lung adenocarci-noma) of 17 patients in the study. Interestingly, 8-Cl-cAMPexhibited a clear biomodulatory effect, thought to be benefi-cial for activation of the immune system. Interleukin (IL)-2areceptor expression, natural killer (NK) cell number, and thecytolytic activity of peripheral blood lymphocytes were sig-nificantly increased, even at the lowest doses of 8-Cl-cAMPadministration (0.01 mg/kg/hr).

The pharmacokinetics, tumor disposition, and metabo-lism of 8-Cl-cAMP was examined in 7 breast cancer pa-tients receiving 0.022 or 0.045 mg/kg/hr as continuous infu-sions for 28 days (Cummings et al., 1996). The steady-stateplasma levels of 8-Cl-cAMP were in the low micromolarrange (0.15–0.72 mM) and 8-Cl-adenosine was not detected.However, the situation changed completely when analyzing3 tumor biopsies of this cohort of patients. In cytosolic ex-tracts of these tumors, 8-Cl-cAMP was not detectable, but8-Cl-adenosine was found in 2 out of 3 biopsies in high con-centrations (1.33 and 2.02 mM). This is in agreement withthe finding that human plasma has only limited enzymaticactivity to hydrolyze 8-Cl-cAMP (Van Lookeren Campagneet al., 1991; Yokozaki et al., 1993; Juranic et al., 1998). Inthe analyzed breast tumors, 8-Cl-cAMP was extensivelymetabolized to 8-Cl-adenosine, indicating an important rolein vivo for 8-Cl-adenosine for the antitumor activity of 8-Cl-cAMP. These findings were supported by the analysis ofHT29 human colon cancer xenografts in mice, which dem-onstrated high metabolic activity and formation of 8-Cl-ade-nosine. This study again claimed the prodrug character of 8-Cl-cAMP in the treatment of solid tumors. Nevertheless, thedata of the Phase I clinical trials have demonstrated the possi-bility of safe administration of 8-Cl-cAMP if hypercalcaemiacan be controlled.

Langdon and co-workers (1998) studied the antitumor ac-tivity of 8-Cl-cAMP against a broad spectrum of human tumorxenografts, namely, HT 29 colorectal, ZR-75-1 breast, HOX60 and PE04 ovarian, and PANC-1 pancreatic carcinoma,implanted in nude mice for identification of potential PhaseII targets. Continuous infusions of the drug strongly inhibitedthe growth of all tumors, but produced hypercalcaemia andmarked loss of body weight. To prevent hypercalcaemia,salmon calcitonin was co-administered with 8-Cl-cAMP tomice bearing the HT 29 colorectal carcinoma xenograft.Salmon calcitonin usually can decrease plasma calcium con-centrations in patients with malignancy-associated hypercal-caemia (Thiebaud et al., 1987). This schedule produced onlymoderately increased plasma calcium levels in some of thetreated animals, combined with equipotent antitumor activ-ity. However, further in vivo studies with the combination ofsalmon calcitonin and 8-Cl-cAMP are required to validatethe efficacy of this therapeutic approach.

In an ex vivo Phase II trial, 8-Cl-cAMP was reported toexhibit growth inhibition of several tumor types from pa-tients who had previously received various chemotherapeu-tic agents (Bosanquet et al., 1997). Acute myeloid leuke-mia, chronic lymphocytic leukemia, and non-Hodgkin’slymphoma reacted most significantly.

8-Cl-cAMP has also been tested in combination withstandard therapeutics against cancer, because it was re-ported to be a novel modulator of multidrug resistance(MDR), one of the main obstacles in current antitumor ther-apy (Yokozaki et al., 1993; Srivastava et al., 1996). Theterm MDR stands for the inherent ability of cancer cells todevelop cross-resistances to a wide variety of structurally


unrelated natural product anticancer agents after exposureof the cells to a single hydrophobic drug, often leading to ul-timate failure of chemotherapy. The transcriptional regula-tion of the MDR-1 gene encoding the P-glycoprotein (amultifunctional drug exporter), as well as its post-transcrip-tional phosphorylation, is under the control of PKA (andPKC), and 8-Cl-cAMP was found to reverse multidrug re-sistance by down-regulation of P-glycoprotein expression(Scala et al., 1995).

Low survival rates in the treatment of advanced or recur-rent cancers call for development of new chemotherapeutictools and new combinations of chemotherapeutics. In thiscontext, 8-Cl-cAMP was tested in combination with pacli-taxel, one of the first-line drugs against recurrent ovariancarcinomas (Einzig et al., 1992), against a multitude of hu-man cancer cells, including ovary, head, colon, lung carci-nomas, and melanomas (Di Isernia et al., 1996; Tortora etal., 1997; McDaid & Johnston, 1999), and in vivo againsthuman colon cancer xenografts in mice (Tortora et al.,1997). The effect of both drugs was either additive (Di Iser-nia et al., 1996) or, in most cases, synergistic (Tortora et al.,1997; McDaid & Johnston, 1999). The synergism was high-est in protocols with paclitaxel application prior to 8-Cl-cAMP. Cell-cycle analysis of several cell lines demon-strated accumulation of cells in the G2/M phase and a highlyincreased level of apoptotic cells. This synergistic effectwas reproduced in the xenograft-bearing mouse model. Be-side paclitaxel, studies with docetaxel, cisplatin, carboplatin,and different retinoic acid derivatives have been conductedto test the effectiveness of 8-Cl-cAMP in combination withcurrent standard chemotherapeutics (Tortora et al., 1997;Srivastava et al., 1998b, 1999).

The promising results of synergistic antitumor activity inthese studies suggest new approaches with combination regi-mens against malignant diseases, which might improve theeradication rates and minimize undesired side effects of fu-ture antitumor therapies. Although 8-Cl-cAMP is currentlyunder investigation as a single chemotherapeutic agent inclinical Phase II trials, it appears likely that further trials willuse 8-Cl-cAMP in combination with other antitumor drugs.

In a recently published additional Phase I study with 32patients exhibiting refractory malignancies (Propper et al.,1999), some earlier clinical data were confirmed, and a new3-day intermittent weekly application regimen of 8-Cl-cAMP in high doses (0.11 mg/kg/hr) was suggested for thecombination of conventional chemotherapy with 8-Cl-cAMP in future Phase II trials.

4.2. Other cyclic nucleotide analogs and growth inhibition

Besides 8-Cl-cAMP, only Bt2-cAMP was tested in moredetail as an anticancer agent. Bt2-cAMP was the first cAMPanalog with relevant growth inhibitory properties, and re-sults obtained with this compound in cell culture and in ani-mal studies are reviewed elsewhere (see Cho-Chung et al.,1991, 1995; Cho-Chung & Clair, 1993). With identification

of 8-Cl-cAMP as a significantly more potent inhibitor of tu-mor cell growth, studies with Bt2-cAMP were discontinued.However, some cAMP analogs were tested together with 8-Cl-cAMP and were shown to produce similar effects, e.g.,8-amino-cAMP (Bøe et al., 1995; Vintermyr et al., 1995).Numerous cAMP analogs were tested in different leukemiacell lines for their ability to induce apoptosis and growth in-hibition. Besides 8-Cl-cAMP, other cyclic nucleotides ex-hibited significant antiproliferative potencies, e.g., 8-amino-cAMP, 7-deaza-cAMP, and 2-Cl-cAMP. Additionally, therole of nucleoside and 59-monophosphate metabolites of pa-rental cyclic nucleotides in the inhibition of cell growth wasinvestigated. With most cyclic nucleotide analogs (including8-Cl-cAMP), the corresponding nucleoside metabolites werefound to produce strong antiproliferative effects (Lanotte etal., 1991; Ruchaud et al., 1995; Hoffmann et al., 1996).

Studies with phosphorothioate-modified analogs ofcAMP in tumor cell growth inhibition are scarce. In a hu-man prostate carcinoma cell line (PC-3-M), Sp-cAMPS in-duced growth arrest with an IC50 of 33 mM, a 15-fold lowerconcentration when compared with Bt2-cAMP (IC50, 500mM; Bang et al., 1994). Some other Sp-cAMPS analogsproduced growth inhibition in Brown Norway rat leukemia(BNS) and rat glioma cells (C6) (Genieser et al., 1992). Sp-5,6-Cl2-cBIMPS exhibited the highest activity (IC50s, 80mM for BNS and 200 mM for C6). In v-abl oncogene trans-formed NIH 3T3 cells, Sp-5,6-Cl2-cBIMPS was as potent as8-Cl-cAMP in induction of apoptosis (Weissinger et al.,1997). In HL-60 leukemia cells, Sp-8-Cl-cAMPS and Sp-8-Br-cAMPS produced growth inhibition at 10-fold higherconcentrations relative to 8-Cl-cAMP (IC50s, 8 mM, 3 mM,0.4 mM, respectively; Yokozaki et al., 1992). Interestingly,Rp-8-Cl-cAMPS (IC50, 3 mM) was as potent as the Sp ana-logs in this study. In summary, due to sparse data, a benefi-cial role for cAMP analogs (with the exception of 8-Cl-cAMP) in growth control cannot be ascribed.

4.3. Cyclic nucleotide analogs in organ transplantation

Recent surgical and immunological advances are the ba-sis for the growing importance of transplantation as the lastoption for patients with end-stage organ diseases. One of themajor problems in transplantation is duration of time for de-livery of organs from donor to recipient. This is especiallycritical in the case of the heart and lung, which are most vul-nerable to fatal injuries due to ischemia, preservation, andreperfusion. Currently, the duration of heart and lung pres-ervation is limited to 4–6 hr because these patients stronglydepend on immediate graft function, compared with otherorgans (Kirk et al., 1993; Young et al., 1994). It is well-doc-umented that progressive dysfunction of the vasculature inorgans during ischemia and preservation is one importantlimitation of organ function after transplantation (Lefer etal., 1991; Pinsky et al., 1993). The vasculature becomesmore adhesive for neutrophil accumulation (Pillai et al.,1990; Adkins & Taylor, 1990), exhibits increased perme-


ability of the cell membranes (Ogawa et al., 1990; Pinsky &Stern, 1994), becomes vasoconstrictive and prothrombotic(Pinsky et al., 1994a, 1994b), and secretes pro-inflamma-tory cytokines such as IL-1a and -8 (Pinsky et al., 1994b;Pinsky, 1995). Prolonged preservation leads to decreases incardiac and lung cAMP levels, while NO and subsequently,cGMP levels decrease during the reperfusion period. In con-trast, elevated endogenous concentrations of cAMP andcGMP have been correlated with prolonged integrity andfunction of endothelial and smooth muscle cells (see Pin-sky, 1995; Eigenthaler et al., 1999). The role of cAMP andcGMP analogs as additives to improve organ preservationfor transplantation by maintaining the vascular function hasbeen studied by several groups. cAMP analogs were foundto increase the barrier function of endothelial cells in culture(Ogawa et al., 1992), and ischemia-reperfusion increasedmicrovascular permeability is prevented by increased en-dogenous cAMP levels in a perfused lung model (Seibert etal., 1992). Pinsky et al. (1993) tested Bt2-cAMP and 8-Br-cAMP as supplements of preservation solutions for cardiacstorage in a rat model system. After a 12-hr preservation,7% of control grafts survived (lactated Ringer solution),compared with survival rates of 93% (1–2 mM Bt2-cAMP)and 100% (100 mM 8-Br-cAMP). Addition of Bt2-cAMP (4mM) to University of Wisconsin solution, a clinical stan-dard solution for cardiac preservation (Jeevanandam et al.,1991), improved survival rates after 24 hr from 35% (with-out Bt2-cAMP) to 75% (with Bt2-cAMP). Cardiac graftsstored with Bt2-cAMP additionally exhibited improvedblood flow and decreased neutrophil infiltration after trans-plantation. Nitroglycerin and 8-Br-cGMP (0.5 mM), addi-tives that trigger the NO/cGMP pathway, were shown to di-minish leukostasis and increase blood flow in cardiac grafts(Pinsky et al., 1994b). The results of both studies were com-bined with development of a new preservation solution,namely Columbia University solution with Bt2-cAMP (2mM) and nitroglycerine (100 mg/L), which exhibited supe-rior survival rates compared with University of Wisconsinsolution in a primate heart transplant system (Oz et al.,1993).

Beneficial effects for graft survival in a rat model forlung transplantation were reported for 8-Br-cGMP (Pinskyet al., 1994a) and Bt2-cAMP or 8-Br-cAMP (Naka et al.,1996) in terms of increased blood flow, improved arterialoxygenation, reduced vascular resistance, and decreasedneutrophil infiltration. A maximum effect was observedwith 0.5 mM 8-Br-cGMP, 2 mM Bt2-cAMP, or 0.1 mM 8-Br-cAMP supplemented to Euro-Collins solution, the clinicalstandard for human lung preservation (Hopkinson et al.,1998). While Euro-Collins solution alone produced z20%graft survival after organ storage for 6 hr, addition of eithercyclic nucleotide improved the survival rate to 80–100%.Under similar conditions, 250 mM 8-pCPT-cGMP was re-ported to produce 100% recipient survival, whereas inhaledNO during the reperfusion period had no protective effecton graft function (Naka et al., 1995). This is in agreement

with reports from other laboratories. For instance, Bt2-cAMP-supplemented perfusion solutions improved lunggraft protection in three studies (Chiang et al., 1997, 1998;Nakamura et al., 1997) and reduced tissue injury after smallbowel preservation (Minor & Isselhard, 1998). 8-Br-cAMPand 8-Br-cGMP were studied in a rat model for liver trans-plantation and found to exert the highest organ protection at0.5 mM each. Survival rates 10 days after transplantationwere between 40% and 100%, depending on the experimen-tal setting (Maeda et al., 1998).

A recently published study compared Columbia Univer-sity solution (with and without 2 mM Bt2-cAMP and 100mg/L nitroglycerin) with Euro-Collins solution, Universityof Wisconsin solution, and low-potassium dextran glucosesolution for preservation in an orthotopic rat model for lungtransplantation (Kayano et al., 1999). After preservation oflungs for 6 hr and transplantation, pulmonary arterial pres-sure, pulmonary vascular resistance (PVR), neutrophil ac-cumulation, tumor necrosis factor-a levels, IL-1a levels,and recipient survival 30 min after transplantation weremonitored for each preservation solution. Columbia Univer-sity solution exhibited superior effects on graft function,with respect to recipient survival rate of 100% (comparedwith 20–50% survival for the others), combined with high-est arterial oxygenation, lowest vascular resistance, reducedleukostasis, lowest levels of IL-1a, and attenuated tumornecrosis factor-a levels. Given the fact that Euro-Collins so-lution is used in 77% of all lung transplantation centersworldwide, followed by University of Wisconsin solution(14% worldwide; Hopkinson et al., 1998), survival rates of37% and 50%, respectively, in this study point to the needof further preclinical trials with larger animals.

Bt2-cAMP and nitroglycerin are not ideal candidates totrigger PKA and PKG signaling in intact tissues. In the caseof Bt2-cAMP, 8-Br-cAMP exhibits comparable effects atsignificantly lower concentrations. To our knowledge, nodata on the kinetics of Bt-cAMP formation and potentialdegradation in preserved organs are available. There may bea link between Bt-cAMP and cGMP degradation and graftfailure. Even with Columbia University solution, recipientsurvival rates decreased significantly with prolonged pres-ervation time from 100% (6 hr), 80% (9 hr), to 30% (12 hr)in the study of Kayano et al. (1999). Therefore, this ap-proach is not a real breakthrough with respect to the criticaltime constraints for donor/recipient organ delivery. Onecould speculate that hydrolysis-resistant lipophilic cAMPand cGMP analogs with improved agonistic properties to-ward PKA and PKG might be better candidates as supple-ments for future preservation solutions. In this context, es-pecially those analogs that exhibit no PKA/PKG specificity,such as 8-pCPT-cAMP, could be of interest here (Section3.2.1). Nitroglycerin, with its induction of the NO/cGMPpathway, is well accepted as being beneficial for vascularfunction. However, NO effects independent of cGMP for-mation and PKG are established (e.g., Eigenthaler et al.,1999). NO-induced formation of toxic peroxynitrite and hy-


droxyl radicals might occur during reperfusion, with poten-tially negative effects on graft function (Hogg et al., 1992;Beckman et al., 1994). Given the fact that cGMP is hydro-lyzed rapidly by endogenous PDEs, cyclic nucleotide ana-logs, such as 8-pCPT-cGMP, with high lipophilicity andsufficient activation potential for PKG, might be improvedadditives for future preservation solutions.

4.4. Pulmonary hypertension or asthma

In the diseased state of pulmonary hypertension or PVRconnected to asthma, standard therapy involves application ofb-adrenergic agonists in spray form (leading to elevated cAMPin the recipient cells). The problem with this kind of therapy isthe occurrence of a down-regulation of the b-adrenergic recep-tor, which brings the patient into a state of resistance. The sec-ond problem is that most, if not all, substances useful for thetreatment of PVR also reduce the systemic vascular resistance,accompanied by cardiovascular problems with prolonged treat-ment. Finally, in patients with a long-standing asthmatic his-tory, even dose-elevation can no longer compensate for thedown-regulation processes mentioned above.

For the latter patients, and for some of those characterizedby the two issues before, new strategies have been developed.Lawson et al. (1995) describe the use of inhaled 8-Br-cGMPin a porcine model of PVR. In their patent documents, they ex-tend this study by patenting the use of inhaled agonistic cAMPand cGMP derivatives (and also PDE inhibitors, NO donors,etc.) for asthmatic conditions (Lawson et al., 1998). To ourknowledge, no clinical studies have been reported. A related,but independently developed, approach has been patented andpublished for the use of an inhaled natriuretic peptide (Urodil-atin) for similar purposes (Flüge et al., 1995, 1999). Both firstmessenger (Urodilatin) and second messenger (cAMP, cGMPetc.) strategies may bring new perspectives for the therapy ofpatients with asthmatic illnesses, especially in the very latestages of the disease.

4.5. Rp-8-bromo-adenosine 39,59-cyclicmonophosphorothioate, a new lead structureagainst diseases of the immune system?

cAMP is established as a negative modulator of B and Tlymphoid cell proliferation (Muraguchi et al., 1984; Klaus-ner et al., 1987; Taskén et al., 1994). The inhibitory regula-tion of T-cell receptor (TCR)/CD3 complex-induced T-cellproliferation by cAMP depends mainly on PKA-I isozymeactivation, as was verified employing suitable cAMP analogpairs preferentially activating PKA-I or -II (Skålhegg et al.,1992). Moreover, PKA-I was shown to co-localize with theTCR/CD3 complex during activation and capping of T-cells(Skålhegg et al., 1994), and TCR/CD3 complex formationwas found to increase the intracellular cAMP level (Kam-mer et al., 1988). Together, both effects led to the hypothe-sis that the cAMP/PKA-I pathway might be seen as a nega-tive feedback mechanism of normal lymphocyte immuneresponsiveness (Aandahl et al., 1998).

Impaired T-cell function, including defective activationand proliferation, is characteristic for certain virus-infectedpatients, such as common variable immunodeficiency (CVI)and human immunodeficiency virus (HIV). The identifica-tion of Rp-8-Br-cAMPS and Rp-8-Cl-cAMPS as currentlythe most effective antagonists of PKA-I in vitro (in cellspredominantly expressing PKA-I; Gjertsen et al., 1995), aswell as the finding that the RIa/RIIa-ratio is z75%/25% ofthe total R-subunit activity in human T-cells (Skålhegg etal., 1992), prompted Aandahl et al. (1998) to evaluate therole of PKA-I in T-cell dysfunction of HIV-infected pa-tients. Despite almost similar patterns of PKA isozyme ex-pression in CD31 T-cells from infected patients comparedwith healthy donors, a 2-fold increased intracellular concen-tration of cAMP was detected in T-cells from HIV-positivepatients, in agreement with the finding that HIV proteinscan increase cAMP levels in lymphoid cells (Hofmann et al.,1993; Haraguchi et al., 1995). In addition, 8-pCPT-cAMPinhibited CD31 T-cell proliferation of infected patientsmore effectively, but with reduced cooperativity comparedwith controls, pointing to a contribution of endogenouslyelevated cAMP levels during activation of PKA-I.

The PKA-I antagonist Rp-8-Br-cAMPS restored T-cellproliferation inhibited by the PKA agonist Sp-8-Br-cAMPSin a dose-dependent manner (8 mM–1 mM) from healthy andinfected blood donors. While Rp-8-Br-cAMPS alone did notmodulate T-cell proliferation of healthy donors, this PKA-Iantagonist improved T-cell proliferation of infected donorsup to 2.8-fold. Interestingly, the highest degree of prolifera-tion improvement with Rp-8-Br-cAMPS was observed inCD31 T-cell preparations that responded poorly to TCR/CD3 stimulation. Patients receiving highly active antiretro-viral therapy (indinavir, zidovudine, and lamivudine) stillexhibited impaired T-cell function, which was improved byRp-8-Br-cAMPS in 6 out of 9 cases 1.5- to 2.8-fold.

Taken together, these results strongly indicate that thecAMP/PKA-I pathway contributes to T-cell dysfunction inHIV-infected patients, and that Rp-8-Br-cAMPS, as an an-tagonist of PKA-I, can increase CD31 T-cell proliferation torestore an improved immune status. Furthermore, thesefindings indicate that PKA-I antagonists are potential futuretherapeutics, together with current highly active antiretroviraltreatment regimes against HIV. In this context, Rp-8-Br-cAMPS cannot be recognized yet as a new potential therapeutic.It should be seen rather as a model compound for developmentof new in vivo PKA-I antagonists, based on the fact thateven at 1 mM Rp-8-Br-cAMPS, stimulation of T-cell prolif-eration of HIV patients reached no plateau (Aandahl et al.,1998). Currently, one can only speculate whether a more li-pophilic Rp-cAMPS analog with enhanced cell permeabil-ity and potent antagonistic properties against PKA-I mayfurther improve TCR/CD3-induced proliferation of T-cellsfrom HIV-infected patients. No such Rp-cAMPS analog hasbeen identified (or recognized) to date, and further syntheticwork, as well as careful mechanistic in vitro and in vivostudies, are eagerly awaited.


Similar immunomodulatory effects of 1 mM Rp-8-Br-cAMPS were reported for CVI patients with T-cell dysfunc-tion in a study in which anti-CD3-induced T-cell prolifera-tion was either significantly improved or fully restoredcompared with healthy blood donors (Aukrust et al., 1999).Torgersen et al. (1997) demonstrated involvement of PKA-Iin cAMP-induced inhibition of NK cell cytotoxicity bycAMP analog pairs selective for PKA-I or -II. Rp-8-Br-cAMPS was shown to counteract cAMP-affected inhibitionof NK activity, as in an earlier report on Rp-cAMPS action(Takayama & Sitkovski, 1989). Tortora et al. (1995) dem-onstrated improved NK cell cytotoxicity in the first clinicalPhase I trial of 8-Cl-cAMP, an immunomodulatory effectthat might be correlated with down-regulation of the RI sub-unit and PKA-I (Cho-Chung et al., 1995). In contrast, thestrong immune cell response in patients with the autoim-mune disorder systemic lupus erythematosus recently wasrelated with an impaired cAMP/PKA-I system in T-lym-phocytes from these patients (Kammer et al., 1994). Allthese results indicate a pivotal immunomodulatory role ofthe cAMP/PKA-I system in several human disorders, andstrongly suggest PKA-I as an important target for immuno-modulation with suitable PKA-I antagonists or agonists.

5. Conclusions

After 30 years of synthesis and testing of cAMP/cGMPanalogs in diverse biological systems, including clinicalstudies in recent times, this class of substances is still grow-ing and represents a promising field for future developments.From amongst the first-generation analogs, 8-Cl-cAMP, inparticular, has proven beneficial for cancer patients, and it issuggested as a potential partner for co-treatment with classi-cal chemotherapeutics. Second-generation analogs with im-proved stability, membrane permeability, and target selectiv-ity now closely follow the first-generation analogs in severalfields, even reaching the stage of clinical studies in somecases. The recent development of bioactivatable third-gener-ation analogs, which possess the most beneficial propertiesof the second generation, combined with drastically im-proved transmembrane delivery of cyclic nucleotides into in-tact cells, are promising new tools, but are only at the begin-ning of their characterization. However, the discovery ofputative new cAMP-binding proteins recently extends thetarget complexity, and demands testing of existing analogson these new receptors. Drug design based on these new pro-teins is ready to start, since both the analogs and the system-atic approaches suitable to test them are already present, andrenders this field potentially very attractive for the future.

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