Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors

TiPS - April 1990 [Vol. 221

150 of the drug. The effects on water important will be particular@ where hydrophobic binding is invalved. In assessi:lg hydrophobic effects it is more appropriate to think in terms of overlapping areas, rather than interactions between points. There is a need for a revised model of the interactions between drugs and receptors

which takes account of the reasons why there are exceptions to Pfeiffer’s rule. References I Pfeiffer, C.

C. (1956) Scir~r 124. 29-30 2 Barlow, R. 8. (1971) /. Plrnn~~.Plu~~~ol. 23,9C-97 3 Lehmann, P. A., de Miranda, J. F. R. and Ari&s, E. J. (1976) Prog. Dr~rg Rrs. 18, 101-142

firnary sequence of cyclic nucleotide phos hodiesterase isozymes and the design of selective inhibitors Joseph A. Beavo and David H. Reifsnyder Primary seqzle~tceiuforrnntion hns been reported for more than 25 drfferent nmmunlinn cyclic uucleotide phosplzodiesternses. Moreover, recent observntious suggest that mnn!y of these isozyznes me selectively expressed in LI limited number of cell types. The fuct that nearly all these different phosphodiestercses hnzle unique prinmy sequences in their cntnlytic or regulntory dotnnim and thnt they me often selectively expressed itnplies that it tnny be possible to modulate individunl isozynzes using specific drugs. Joe Beavo and David Reifsnyder summrize much of the evidence that hns led to oldr current understanding of multipIe isozyrnes of phosphodiesterczse, with emphasis on aspects thnt my be relezmt to drug design. They also discuss 7ulry ninny previous attempts to isolate isozyme-selective inhibitors may have failed. Although many texts imply that cyclic nucleotides are degraded by a single enzymatic activity, it is now apparent that inactivation of CAMP and cGMP is catalysed by not one, but rather a large number of different cyclic nucleotide phosphodiesterases. Recent data suggest that at least five different isozyme families exist and more than 20 distinct enzymes are now recognized (see Box). More importantly, biological reasons for this great diversity are beginning to be appreciated. For example, many of the isozymes are differentially expressed and regulated in difI. A. Benoo

is Professor nt

tlte Dcparhwn~ 51-3U. Utllvcrsity of WA 98195. US& atld D. H. Rrifsnydrr is n Scientist at Geueutech, Sm Fraucisco, CA 94080, USA.

of Pharmncolo~y. Wasltiqton, Stvtttc.

ferent cell types. From a pharmacological perspective, just as multiple receptors controlling the synthesis of CAMP and cGMP offer opportunity for selective therapeutic intervention., multiple ‘receptors’ for cyclic nucleotide degradation should offer equally good possibilities. Here we discuss the way in which recent data on the structure, regulation and localization of multiple phosphodiesterases provide a conceptual rationale for the design of selective inhibitors and activators of these enzymes (see Refs 1 and 2 for more general reviews on phosphodiesterases).

Early phosphodiesterase inhibitors Since Butcher

the original reports by and Sutherland3 in 1962

4 Barlow, R. B., Franks, F. M. and Pearson, J. D. M. (1973) /. Med. U&w. 16, 439-446 5 Barlow; R. B. (1973) /. Med. Clte,,r. 16, 1037-1038 6 Barlow, R. 6.. Berry, K. J., Clenl,>n, I’. A. M., Niko!aou, N. M. and Soh, K. S. (1978) Br. 1. Pltarr~mcol. 58, 613-620 7 Barlow. R. B. and Burston, K. N. (1979) Br. /. Plu~n~mcol.66, 581-585 8 Barlow, R. B., Birdsall, N. j. M. and Hulme, E. C. (1979) Br. 1, PJxnr~t~ncol.66, 587-590

that methylxanthines such as caffeine and theophylline inhibit CAMP hydrolysis, many studies have been carried out to identify drugs that inhibit cyclic nucleotide phosphodiesterase activity. In the first 20 years of this period no new therapeutically important agents were identified. Although drugs like papaverine and theophylline were found to be relatively potent and effective competitive inhibitors of CAMP and later cGMP hydrolysis, most new agents based on these structures were either ineffective or tou toxic tu be of widespread clinical use. We now know that the relatively poor therapeutic index of these agents was due at least in part to the fact that they were not isozyme specific and therefore increased cyclic nucleotide levels in many non-target cells. Moreover most had additional modes of action. For example, theophylline and its more potent congener 3-isobutyl-l-methyl xanthine (IBMX) are also potent antagonists at adenosine receptors. Dipyridamole, a classical antithrombotic agent that is now known to be a selective phosphodiesterase inhibitor, is also a potent inhibitor of adenosine transport. It is now evident that the design of most early screening experiments was inappropriate. In nearly all of them, whole tissue homogenates or extracts were used as the source of phosphodiesterase activity. Because most tissues are heterogeneous with respect to cell type and many cells contain multiple phosphodiesterases, the screening studies were all conducted with a mixture of isozymes and were therefore unlikely to identify inhibitors selective for an individual isozyme. For a few agents it was noted that l&20% of the total phosphodiesterase activity was inhibited by relatively low drug

TiPS - April 2990 [Vol. 2 21

As might be expected from the fact that new phosphodiesterase isozymes are continually being identified, nomenclature has not been consistent. Many investigators have defined different ‘forms’ on the basis of the order of elution from DEAE resin, using names such as peaks I, II, III, or IV, often modified by a qualifier describing regulatory or kinetic properties. Unfortunately, many of the ‘peaks’ of activity are now known to contain multiple isozymes. Moreover, the order of elution of isozymes from DEAE varies with species, tissue, pH, eluting salt and in some cases the physiological status of the tissue before fractionation. The classification system used in this article is based largely on primary protein and cDNA sequence information. It is likely that there are at least five distinct but related gene families coding for cyclic nucleotide phosphodiesterases in mammals. Moreover, most of these families contain two or more closely related subfamilies (i.e. very similar but distinct genes) and many of the genes appear to give rise to two or more alternatively spliced mRNAs. Therefore, an individual phosphodiesterase isozyme is named according to both the ‘family’ and the ‘subfamily’ of closely related isozymes to which it appears to belong. As more information becomes available, the numbers of individual subfamilies and perhaps even families may increase. Highly specific probes will be needed in order to determine the family and subfamily of new isozymes. A separate descriptive name and Roman numeral is assigned to each of the five families (see Table). These Roman numerals are arbitrarily designated and do not necessarily correspond to orders of elution from DEAE cellulose chromatography or other fractionation procedures. However, where possible they are similar to many previously used nomenclature systems. In germeral, members of one family share 20-25% sequence identity with members of another family. Much of this identity occurs in one domain thought to be catalytically active. Most families include several subfamilies of more closely related isozymes. A capital letter designates each subfamily of this type. Although incomplete, the data currently available suggest that most individual subfamily members are encoded by different but highly homologous genes (70-90% identity). Finally, many of the subfamilies have multiple members that are likely to be the products of alternative mRNA splicing; these are designated by an Arabic numeral. For example, the 59 kDa Ca’+-calmodulin-dependent phosphodiesterase that is a major tsozyme in bovine heart would be called Ca2+-calmodulin-dependent phosphodiesterase, 1~1. Several of the isozymes have not been studied either BS homogeneous proteins or as expression products and therefore are still rather tentative. In particular several of the subfamily members of the CAMP-specific phosphodiesterases (IV) are only identified as single or even partial cDNA clones. Where primary protein sequence data or multiple independent criteria exist for their expression and identity, isozyme names are given in italic type.

151

Family/subfamily

designation

I Ca* +-calmodulin-dewndent IAl 59 kDa heart isoiyme 1, le,

61 kDa brain isozyme 63 kDa brain isozyme

lc,

67 kDa smooth muscle form Ior 67 kDa ‘low K,,,’ form 1;; 75 kDa brain &MPselerfiw form IF1 75 i&d lung form

II cGMP-stimulated family /I,,, 105 kDa heart/adrenal isozyme II= 105 kDa membranebound form llB, 66 kDa liver form

Ill cGMP-Inhibited family Ill,,, 110 kDa hearVplatelet/ adipocyte isozyme

111~2 105 kDa S49-K3Oa form

IllA 63 kDa dense vesicle form IW CAMP-specific family IV,,

k IV,, IVs-

V cGMP-specIfIc famIly V, 93 kDa lung/platelet isozyrne V,, 99 kDa rod membraneassociated isozyme V,, 99 kDa rod soluble isozyme V,, 99 kDa cone isozyme

Comments

Ref.

familv maybe present in cells other than cardiocytes’ a major isozyme in brain’ partial sequence: a separate gene product from IA no sequence data no sequence data no sequence data

a

:

C

c&f 9

no sequence data; includes tightly bound calmodulin major isozyme in adrenal cortex no sequence data: slightly different peptide map than II*, not yet clear if IIs, is proteolytic product of lIA inhibited in vitro by low concentrations of cGMP; no sequence data not yet clear if this is a ‘mutant’ form of Illnr; no sequence data may be activated by insulin: no sequence data

h

k

I

m

n

originally called RDl an6 0.P rat PDE2’ originally called RD2’ P 0 originally called RD3’ originally called DPD’ originaiiy called z ratPDEC: may be same as I&, originally called ratPDE1 P r originally called ratPDE3.1’. r orlgrnally cawc ratPDE3.2; may be due to an internal deletion in IVD,’ also present in vascular smooth muscle activated by lighttransduciwGTP; [Y.fi and y subunits contains b. (Y.fi and y subunits np p subunit; contains (Y, 5 and a larger form of y subunit

s

t U

v

Product of one of two or more alternatively spliced mFtNAs of subfamily gene. RC, ‘rat dunce-like’: DPD. ‘dunce-like’ phosphodiesterase. ‘Hansen R. S. and Seavo J A. (1982) Pruc. Nat/ Acad.SC;. USA 79. 2788-2792:bSharma, R. K. ef al. (1984) J. Biol.Chem. 2,59.924%9254;5Mutus. 8.

l

pers c&mun:‘“Rosei P.‘ef’a/. (1988) J. No/. Chem. 263. 15521-15527;‘Geremla. R. ef al (1984) Eiochem. J. 217.692-700; Smoake. J. A. efaf. (1981) Arci. 6iochem:Bioph~. 206 331-339 gshenoliker. S. eta/. (1985) Siochemistry24.672-676; “Sharma, R. K. and Wang. J. Ii. (1986) J. Sio/. Chem. 261. 14160-14168,‘Martins. T. J.‘eta/. (1962) J. Viol Chem. 257, 1973-1979;1Yamamoto. T. eta/. (1983) J. Biof.Chem. 258. 1252612533: ‘PYne. N. J. &al. V. M. eta/. (1982) J. Siol. Chem. 257.9349-9355: T986) Biochem. J. 234,325-334: ‘Harrison, S. A. ef al. (1986) Mol. PharmacU/. 29,506514. . mSmtheffis. pSwinnen,J. V. era/. (1989) Pro?. Pyne N. J. eta/. (1987) Biochem.J. 242, 3342; OChen, C. N. era/. (1986) Proc. NaBAcad. Sci. USA 83.9313-9317; Nat/ Aiad, Sci. USA 88 5325-5329. qcolicelli J. etaf. (1989 Proc. NaflAcad. Sci. USA 863599-3603; ‘Swinnen. J. V. era!. (:989) t%c. sc~ nzymol. 159,722_729;‘Baehr, W. eta/. (1979) J. Biol. Chem. 254.11669-11677; “Gillespie, USA 86. 8197-82fJl; skrancis, S. l-i. eta/. (li88) Methods E’ P. G. et a/. (1989) J. Viol. Chem. 264, 12187-l 2193; ” Gille: ie P G and qeavo, J. A. (1986) J. Biol. Chem. 263,8133-8141. See also Beavo.J. A. (1988) Adv. Second Messengers Phosphoprotein Res. 22.1-38 for P, urther”details.

Narl Acad.

TiPS - April 1990 [Vol. 121

152

I

? WI

1-1 catalytic domain

calmodulin binding domain

?

I

~~,;;l;calmoduR-dependenl

I

I

1-1

1-1 cms&ygillQ

cps;

?

I

cGMP-stimulated family

I

CAMP-specific family

I

It------rl caaiftiti

1

7

cGMP-specific family

I

I

Ic-----rl cGz;$$ting

i-1 p$;

Fg. 1. Structual ff3afiire.sofmarnmalkn phosphodi~tefase isozyme families.

even though the I& was quite high. Such data are now often reinterpreted to indicate that the test compound was a good selective inhibitor of a phosphodiesterase isozyme that contributed 10-20s of the total activity and a poor inhibitor of the other isozymes in the sample. It is now clear that kinetically pure preparations of individual isozymes must be used in screening for isozyrne-selective inhibitors. concentrations

Primary sequence comparisons Until 1986 the rationale for developing isozyme-selective phosphodiesterase inhibitors was based largely on a combination of faith and empirical trial and error. More recently a firm structural basis has been developed. The first comparative primary sequence information was reported in late 19% for four different cyclic nucleotide phosphodiesterases: the 61 kDa Ca2+-c~m~ulin-dependent isozyme IAX; the cGMP-stimulated isozyme 11~~;and CAMP-specific isozymes from yeast and Drosopf~iln~. It is clear that each of these phosphodiesterases is encoded by a distinct gene. However, one region is highly homologous m all four genes despite the fact that they were all isolated from different species and encoded enzymes with different kinetic properties.

Sequence information has now been obtained for another 11 mammalian phosphodiesterases from several isozyme families: l cDNA sequences for low K, CAMP-specific phosphodiesterases IV,, IV,, IV= and IVo from rat brain7,s and testis9; l cDNA sequences for the photoreceptor cGMP-specific phosphodiesterase (x subunit Vsrru from bovine rod19 and IY’ subunit Vcl= from bovine cone phosphodiesteraserr*r2; l partial sequence for cGMPstimulated phosphodiesterase 11~1 from bovine adrenal gland12; l complete protein sequences for the 61 kDa Caz+~almodulindependent phosphodiesterase 1,~ from bovine brain and the cGMPstimulated phosphodiesterase 11~1 from bovine heart, and partial peptide sequences for the 59 kDa and 53 kDa Ca2+-calmodulindependent phosphodiesterases IAl and 1~s (II. Charbonneau et al., unpublished); l partial amino acid sequence of cGMP-specific phosphodiesterase VA1 from lung13. These isozymes aii contain a homologous region in the C-terminal part of the molecule that is now known to be part of the catalytic domairW4. All the isozymes so far sequenced that

contain high affinity non-catalytic cGMP binding sites** (e.g. the cGMP-stimulated 11~1, and the photoreceptor Va and Vc isozymes) also contain a second homologous domain; this is thought to contain the cGMP-binding site(s) (Fig. I). From a drug development viewpoint, the similarities of the catalytic domains provide a probable explanation of why most early phosphodiesterase inhibitors were not selective. More importantly, the differences in sequence among the catalytic and non-catalytic domains provide a solid experimental basis for the development of sctlective inhibitors for individual isozymes. cDNA sequences have been determined for two different nonmammalian nucleotide cyclic phosphodiesterases that are homologous to each other but do not contain the highly conserved C-terminal region present in mammalian enzymes. The first of these isozymes is one of two cyclic nucleotide phosphodiesterases in yeast6 and the other is the major CAMP phosphodiesterase in the slime mold DictyosteIium discoiditmls. Tc date no functional homolog of either of these enzymes has been found in mammalian systems. Identification and localization of isozymes In order to design isozymeselective phosphodiesterase inhibitors as therapeutic agents for specific diseases, and to predict and avoid side-effects, it is necessary to know the cellular and subcellular distribution of each isozyme. However, understanding of the localization of isozymes has lagged far behind our knowledge of their structural properties. In no case has the complete repertoire of phosphodiesterase families expressed in any given tissue been determined. Even less is known about which isozymes are expressed in which cell type. Since an individual isozyme can be highly localized in a minor cell type of a tissue (see below), most tissue fractionation schemes have not given definitive answers. It will become simpler to address this question as more primary sequences become available and isozyme-specific corresponding nucleotide and immunological probes are designed.

TiPS - April 1990 [Vol. I I] In early studies, histochemical methods based on lead precipitation of the reaction product indicated that high phosphodiesterase activity is localized in postsynaptic endingsib. Although this approach is limited in its ability to discern individual isozymes, some information was gained by carrying out the reactions in the presence and absence of Ca*+-calmodulin. More recently, use of antisera to Ca2+calmodulin-dependent phosphodiesterase(s) has demonstrated that the 61 kDa Caz+-calmodulindependent isozyme is present in high concentrations in the dendritic fields of Purkinje cells and in the pyramidal cells of the cerebral cortex”. There are few other localization data available for this family of isozymes. Brunton and colleaguesi have reported that the major Ca2+-calmodulin-dependent phosphodiesterase in heart, IAl, is not present in cardiocytes. This implies that, since the isozyme is relatively abundant in heart, it must be present at higher concentrations in some ‘minor’ cell type of unknown identity. Arianoi9 has shown, using antisera selective for the cGMP-stimulated phosphodiesterases, II, that this isozyme family is widely distributed in the CNS. Most cells staining for this isozyme also contain relatively high concentrations of cGMP-dependent protein kinase. More recently MacFarland et nl. have found very high concentrations of the same isozyme in the glomerulosa cells of the adrenal cortex where it probably plays an important role in regulation of steroidogenesis by atria1 natriuretic peptide (see Ref. 1 for discussion). The glomerulosa cells make up a relatively small percentage of the adrenal gland, and the phosphodiesterase concentration may approach or exceed the steady-state level of CAMP in these cells. A picture for distribution of the cGMP-inhibited phosphodiesterase family, III, is even less well developed. This family is present as a major component in heart, some smooth muscle, platelets, adipocytes and liver. HOWever, this distribution has been inferred largely from inhibitor studies and DEAE fractionation characteristics, and it is not known how many members of this family

153 exist. It is likely that members of this family are present in other cell types. The CAMP-specific phosphodiesterases, IV, are thought to be more widespread. They are abundant in the CNS and in the reproductive system, and the enzyme was originally studied in kidney and lymphocytes20. Very little is known about the isozyme subfamily distribution, but Conti et e/.zi have shown that the members are differentially expressed in different cell types of the testis and that the expression of at least one can be induced by CAMP. Finally the distribution of the cGMP-specific phosphodiesterases, V, is best characterized in the retina. The earliest localieation data for the cGMP-specific photoreceptor isozymes22 indicated high concentrations of the enzyme in outer segments. More recent data indicate that there are separate isozymes in the rod and cone photoreceptor outer segments23. The concentrations here are probably the highest for any phosphodiesterase in any cell type and may approach X-30 PM. The localization of the non-photoreceptor subfamily of cGMP-specific phosphodiesterases, VA, in other tissues is only now beginning to be determined. Tissue fractionation data indicate that it is abundant in platelets and lung tissue24Js, although it is not known which cell type(s) in the lung express the isozyme. This isozyme also appears to contribute a major component of cGMP hydrolysis in vascular smooth muscle. Although there is very little information on localization of individual phosphodiesterase isozymes at the cellular level in complex tissues, several isolated cell types have been examined. For example, platelets contain at least three different isozymes26 and probably low levels of two others, sti that all the isozyme families are represented. On the other hand mature mammalian red blood cells do not contain phosphodiesterase activity. In adipocytes the predominant isozymes are of the cGMP-inhibited family, III (Ref. 27). Cardiocytes appear to contain mainly cGMP-inhibited and cGMPstimulated isozymes, although the whole heart also contains a calmodulin-dependent isozyme’*. A few cells appear to contain

only one isozyme. For example, the predominant phosphodiesterase in murine S49 lymphoma cells is of the low K, CAMPspecific family, IV (the subfamily is not known). Moreover, ihe relevance of this data to native lymphocytes is not clear since phosphodiesterase gene expression is probably altered in transformed cell lines. Sensitive and selective probes will be required in order to determine n;:jich isozyme families, and which :am& anci :Zti:;famil, members, are prz;ent in individual cell types oi complex tissues.

Pharmacological implications of phosphodiesterase regulation

Studies from many different laboratories indicate that most phosphodiesterase isozymes are subject to strong regulatory influences in the cell. Moreover, different mechanisms often apply to different isozymes. These include feedback control of activity by several different protein kinases2*J9 as well as direct control by the intracellular second messengers Caz+ and cGMP (see Ref. 2 for detailed discussion of regulation of individual isozymes). Phosphodiesterase activity can in theory, therefore, be modulated not only by direct binding of drugs to the enzyme but also by pharmacological modulation of the normal physiological regulatory pathway. For example, since natural regulators like Caz+-calmodulin or cGMP can precisely and selectively regulate different phosphodiesterases, there seems to be potential for producing isozymeselective drugs that mimic or antagonize these regulators. Such agents would be expected to be much less toxic than the relatively nonselective ‘first-generation’ phosinhibitors. For phodiesterase example, a cell-permeable nucleotide analog that mimicked the effect of cGMP on both the cGMPstimulated and the cGMP-inhibited phosphodiesterases might cause minimal effects on cardiac tissues since both these isozymes are present in these tissues. However, it would be expected to lower cAMp concentrations in the adrenal cortex or the liver where the cGMP-stimulated isozymes are predominant, and would be expected to increase CAMP concentrations in smooth muscle or

TiPS - April 2990 /Vol. 221

15-l TABLE 1.inhibitors selective for phosphodiesterase isozyme families toezyme family Drug

l&r (VUJ

Cez ’ -calmodulin-dependent Phenothiazines (e.g. TFP) v~n~tine (TCV-38) 8-metho~methyl-3-iSObu~l-lmethylxanthine

20 4

Original source

SmithKkne Beckman Takeda Chemical Industry Welts ef al.

Ref.

a b C

cGMP-atimulated None known cG~~nhi~~ Cilosfamide (DPC3689) Milrinone (WIN47203) Enoximone (MDLl7043) lmazodan (Cl9141 SKFQ-4120 Trequinsin (HL725) Quasinone (Ro136438) lndolidan (LYl95115) Carbazeran Anagrelide (BL4: 62A)

Otsuka Stirling Winthrop Merrill Dow Warner-Lamberl SmithKline/French Hoechst Hoffmann-La Roche Lilly Pfizer Bristol Myers

B

2

Hoffmann-La Roche Schering AG

m n

0.9 0.8

LederIe(Sigma) May & Baker

9

0.005 0.3 6 11 0.0603 0.6 0.08

CAMP-specific R0201724 Rolipram (ZK62711) cGMF%peciflc ~p~d~ole Zaprinas: ;&f,:aB22946;

i.05

Common nonselectlve lnhibltors -200’ Searle (Sigma) Theophylline -2-50’ 3-lsobutyl-l-melhylxanthine Searle (Sigma) Paoavedne -5-2s Boehrlnaer fSiama1

k

0

: r

This Table is not meant to be complete and in some cases not all possible specificity assays have been carried out. Most agents show at least ZO-foldselectivity for the family listed. ICsOs are for s 1 HIMsubstrate. SKF94120: ii-(4-acetamidophenyl)pyrazin-1 H-one ‘May also inhibit cGMP-specific isozymes with similar affinity: * lCiiO depends on isozyme beingtested.a Levin,Ft.M. andWeiss, B. (1977) A&f.Pharmacot.13,698-697; b Hagiwara. M. eta/. (1984) Biochem. Phannacol. 33.453-457: EWells, J. N. and Miller, J. R. (1988) Methods fnzpwi. 159, 489-4g6; dHidaka, H. and Endo. T. (1984) Adv. Cyc/ic Nucleafide Res. 16, 245-259; ‘Harrison, S. A. eta/. (1986) Mol. Pfwmacol. 29.506514; ‘Kariya T. eta/. (1982) J Car&w%. Ffwmaw! 4, 509-514; s Weishaar, FL E. ei al. (1988) Biocbem. Fharmaco!. 35.787-890; h Simpson, A. W. ef al. ( I986) &r&tern. F~atma~i. 37,23152320; ’ Ruppert. D. and Weithman, K. U. (1982) Life Sci. 31.2037-2043; i Ho&k, M. et al. (1984) J. Cardiovasc. Pharmacol. 6. 520430: ‘Kauffman, R. F. et al. (1987) Mol. Pharmacul. 30. 609-616; ‘Gillespie, E. (1988) Biochem. Fharmacol. 37, 2866-2868: mSheppard, H. et al. (1972) Adv. tick Nuckotide Res. 1, 103-l 12; “Schneider, H. H. et a/. (1986) Eur. J. Phatmacol. 127, 105-l 15;oGillespie. P. G. and Beavo, J. A. (!989] &fo/. o&rnrncol. 36, 773-783; PBu:cher, R. W. and Suther!and, 5. W. (i962) .f. Biol.Cfiem. 237, I %4--l 25C; 9 Beavo, J. A. et al. (1970) A@. F!?am?acol.6.597-603; ‘Kukovetz. W. R. and Poch. G. (1970) NaunpSchmiedeberg’s Arch. Pharmacol. 267,189-l 94.

where the AMP-inhibited isozyrne is thought to predominate. On the other hand, a drug that altered the activity of only one of these isozymes would be expected to have a different pharmacological profile. Perhaps the best examples of selective inhibitors are the phosphodiesterase inhibitor cardiotonic drugs such as milrinone and amrinone (see Table I). In heart, they increase the force of contraction, presumably by increasing Ca2+ levels secondary to increilsing phosphorylation of a cardiac U+ channel. This effect is mediated by inhibition of the IMP-inhibited phosphodiesterase, III. Since the effects of platelets,

the drugs on phosphodiesterase III are inhibitory and quite selective they dre more pharmacologically effective than cGMP itself. Nearly all of the cardiotonic drugs are also good smooth muscle relaxants and platelet aggregation inhibitors. In these tissues, the effects of a selective inhibitor of the phosphodiesterase II1 family would be expected to mimic more closely those of cGMP since little of the cGMPstimulated isozyme family, 11, is present. Indeed, increases in &Ml’ concentration elicited by agents like EDRF have large effects on smooth muscle relaxation and platelet aggregation, particularly when acting in conjunction

with drugs that stimulate adenylyl cyclase. The classical observation that phosphodiesterase inhibitors have greater effects on cyclic nucleotide levels when adenylyl or guanyiyl cyclase is stimulated should provide an additional opportunity for highly cell-type-selective intervention. Submaximal doses of a selective cyclase agonist along with an isozyme-selective phosphodiesterase inhibitor (which presumably would also be at least partially crll-type selective) should cause the greatest changes in cyclic nucleotide levels only in those cells containing both the receptor for the cyclase agonist and the specific phosphodiesterase inhibited. As a result therapeutic efficacy should be increased and side-effects decreased. It may be possible to design a molecule containing both activities. Agents working via other pathways that act synergistically with CAMP or cGMP would also be possible candidates to be used with selective phosphodiesterase inhibitors. Selective inhibitors currently available A number of selective inhibitors of various phosphodiesterase isozyme families have been described, some of which are beginning to be used both experimentally and in the clinic (Table I). However, to date no inhibitor distinguishing between members of the same kiZiiiy has been reported. All these agents exhibit at least 20-fold selectivity as inhibitors of a particular isozyme family. The largest group is those that inhibit the cGMP-inhibited phosphodiesterases, III, and many of the published data concern these compounds. Interestingly, a relatively wide range of structures are able to function as selective inhibitors of this isozyme family (Table I lists only a few representative examples). Many of these compounds are used as cardiotonic and antithrombotic agents as well as vasodilators [for reviews, see Refs 33 and 34, and B. Wetzel and N. Hauel (1988) TiPS 9, 166170]. There is also substantial interest in using selective phos-

phodiesterase inhibitors as antrthrombotic agents, vascular and aitway smooth muscle relaxants, anti-in~ammatory agents and antidepressants.

TirS - April 2990 [Vol. 2 I/ Many fewer selective Inhibitors are available for the other phosphodiesterase isozyme families, such as the Caz+-calmodulin family. Many of the ‘anti-calmodulin’ drugs, such as trifluoperazine, do of course inhibit isozymes of this family selectively. However, they also inhibit nearly all other calmodulin-dependent enzymes and most also bind to other proteins in the cell. This lack of overall selectivity severely limits their use and increases their side-effects. B-methoxyVinpocetine and methyl-3-isobutyl-l-methyl xanthine appear to be relatively specific for the Ca2+-calmodulindependent phosphodiesterases as they appear to inhibit the cata!ytic rather than the ca!modulin binding site (Table I). Very recently, Hidaka’s group reported35 an agent that may act by selectively binding to the calmodulin binding domain of CazT-calmodulin kinase II and selectively inhibit activation of tl is enzyme (i.e. it does not inhibit Ca2+-calmodulin-dependent phosphodiesterase). If analogous agents can be found for the Cal’ -calmodulindependent phosphodiesterases, then it should be possible to distinguish members of this isozyme family pharmacologically. A notable absence from Table I is selective inhibitors of the cGMPstimulated phosphodiesterases, !I,+ To our knowledge there are no isozyme-selective inhibitors of this family. Low concentrations of many other phosphodiesterase inhibitors actually stimulate this isozyme activity34 - a property that greatly increases the difficulty of finding a selective inhibitor. In fact, activators of this isozyme might prove to be valuable in decreasing Ca2+ conductance in the heart, aldosterone synthesis in the adrenal gland, and possibly neurotransmitter release, since stimulation by cGMP of phosphodiesterase II* appears to cause similar effects in intact cells1,2fl,30. It should be possible to achieve such selectivity since there are differences between the ligand specificity of the non-catalytic allosteric and catalytic sites on this phosphodiesterase and those of other isozymes. It may also be feasible to find activators of the other phosphodiesterases and test their effects in vim. Dipyridamole and zaprinast are

155 effective, selective inhibitors of the cGMP-specific phosphodiesterases VI\ and Vs. In some early studies it was reported that these agents will also inhibit Caz+calmodulin-dependent isozymes. It now seems likely, however, that many of these results were due to cGMP-specific phosphodiesterase contamination of the preparations being used. Dipyridamole would be expected to increase cGMP concentrations in selected tissues and it has been used extensively in the clinic, usually in combination with aspirin as an antithrombotic agent. However, it is not yet clear how much of the efficacy of this mixed drug treatment is due to cGMPspecific phosphodiesterase inhibition. Recently, both zaprinast and dipyridamole have also been reported to be potent and selective inhibitors of the photoreceptor cGMP-specific phosphodiesterase (Table I). 0 0 0 Cyclic nucleotides mediate many different effects in the animal. The differing distributions of phosphodiesterases between cell types provides one way of increasing specificity and regulation of these cyclic-nucleotide-mediated processes. Moreover, the large diversity in structure, function and regulation phosphodiamong esterase isozymes suggests that it will be Fossible to develop selective, and possibly therapeutically useful, inhibitors and acti,/ators of individual isozymes. In order to realize thi- promise, it will be necessary to sci’een cumpounds on kinetically pure prep,arations of individual isozymes. This process will be simplified by the availability of cDNA clones encoding many of these isozymes, and of efficient systems for expressing mammalian proteins in veast strains or mammalian cell lines having very low background phosphodiesterase activity. References Beavo, J. A. (1988) Ado. Serord Mcssrrgm Phosy/qwofcirr Rrs. 22, l-38 Houslay, M. D. and Beavo, 1. A., eds (1990) Isq/rws of Cyclic Nlrrlrofidt* Plrospko~;esfrrns~~s, John Wiley & Sons Butcher, R. W. and Sutherland, E. W. (1962) /. Biol. Clxw. 237, 1244-1250 Charbonneau, H., Beier, N., Walsh, K. A. and Beavo. 1. A. (1986) Proc. N~tl Ad. Sri. USA 8j; 9308-9312 Chen, C. N., Denome, S. and Davis, R. L. (1986) Proc. Nnll A&. Sri. USA

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