TiPS-januay

1991 /Vol. 121

with unknown concentration. Ry contrast, the method presented here can be used to determine the receptor subtype mediating effects of an endogenous agonist which may not necessarilybe the receptor predominantly mediating the effects of the corresponding exogenousagonists.Also, this method may be applicable to any biological system in which the agonist-receptor interaction is studied at the level of the primary stimulus, electrical or chemical, responsible for releasing the agonist. Last but not least, this method allows rapid assessment of receptor types operative in a

13 functional test. A screening method that is more rapid than pA2 determinations is often desirable. References

I Tallarida, R. J., Cowan, A. and Adler, M. W. 11979)Lifr Sn. 25.637-654 2 Kenakin, T. P. b84) Ph&wco/. Rrv. 36, 165-222 3 Black. J, W. and Shankley, N. I’. (1985) Rr. /. Plmnracol. 86,6B1-607 4 Shankley, N. P., Black, J. W., Canellin, C. R. and Mitchell. R. C. (1988) Br. /. Phanrmrol.94,264-274 5 Black, J. W. and Shanktey, N. P. (1986) Er. J. f’hant~acool. 88, 291-297 6 Ciachetti, A., Angelici, 0.. Michcletti, R. and Schiavone, A. (1986) Tnwfs Phnn~Iaco/.Sri. wool.5 Seypl. Sllhryyn of Mrrscnri~rirRccceptors I/) 88

Progressin the development of potent bombesin receptor antagonists Robert T. Jensen and David H. Coy Bombesinand the mammalian-rela!ed prprides gaskin-releasing ycplidc GRPA GRPI,I_~~ and neuromedin B have been shozon to have numerous actions in the CNS, guslrointesrsfinaltract and on g:owlh. However. Urr t-o/c of the peptides in variousphysblo$cal processes has rernnincdunclear brcause of fhc lack of potent anmagonisb. Recent in vitro studies have described four d#?rent classesof bombesin receptor antagonist, some of which are active irr the nanomolar range and in vivo. Robert jensen and David Coy describe recent insights into peptide structural determinantsof biological activity. Evidmce @m structure-function studies have resulted in idenfification of some analogues that function as potent antugonisb in all sysfems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist. The amphibian peptidesbomb&n and iitorin, as well as the structurally related naturally occurring mammalian peptides gastrinreleasing peptide (CRP), GRPI,+2? and neuromedin B (Table I), have been shown to have a wide range of biological or pharmacological R. T. /ensen is Chirf of Ihc CPII Bio/o~y Section. LXptivr DiscaaesBrurrh, NnGonal /nsPuk of Diabetes, Diptivr and Kidq Dbrasfs, a) tkr N&ala/ Imlilrks of ffrrlrh. Bfrhrsda, MD 20692, USA and D. H. Coy,is Pmffsaor of Mbfifiw. Twlata UtriwrafQ Mrdfcal Cetrtrr. Nrw Or/raw. LA 70112. USA.

actions’. These include stimulation of the release of numerous gastrointestinal hormones and peptidesz,stimulation of secretion by various exocrine glandGJ, contraction of smooth muscle’, chemotaxiss, behavioral effecW, CNS effects such as thermoregulation’, increased satietynand the ability to function as a growth factor in 3T3 cells, human small cell lung cancer (SCLC) cells, and normalbronchialepithelialceW-I*. Bombesin-related peptides have been proposed to have an autocrine growth mechanism in regu-

M. and Figala. V. !I9881 Eer. /. Phnrmml. 158, 11-19 Kramer. W.. Baron, E., Boer, R. and E!tze. M. Nasnyn-Schnfthbzrs’s Art+. Phwv~nwf.(in press) Bevan. J. A. and Shrwc. S. M. (1989) TrcvrdsPhareracnl.Sri. IO, 63 Raffa. R. 8.. Vaught, J. L. and Porreca.F. (1989) TrcedsPharetecol.Sci. 10,183-185 Kmmer, W.. Baron. E.. Boer, R. and Eltze, M. (1990) N~Issyn-Srbmirnhcr~‘s Arch. Phmmwo/. 341, 165-170

7 Eltze. 8

9 10 II

AFDX:(ll1[2-lfdiethylamino)methyl)-lpiperidinyljacetyll-S,Il-dihydro-bHpyridol2361[1AJbi~i~-6-)one BTMIO% ris-(-)-2,3-dihydru-3+-mcthylI-piperazinyl m~hyl)-2-phenyi-l,5~nzothiazepin-4fSHJ~onemonohydrochloride 4-DAMP: 4-diphenylacetoxy-N-methylpiperidine methiodide NMS: N-methylscopolamine

lating the growth of SCLC cells, becausebombesin receptorantagonists or anti-bombcsin antibodies inhibit grotith irr vitro and in vivov.13. Potent rcccptor antagonists would bc useful not only to determine the precisc~ physioio$cal importance of bombesin in these various physiological processes, but atso to invcstigatc their clinical ptcntial as inhibitors of the growth-promoting cftcctsof these peptides. Four classesof bombrsin rrrrptor antagonists have been idrntified’4 z2. The first class, dcwribcd in 1984’4, is generallyactive in the micromolar range (0.1-111)IIM)

and includes the o-amino acidsubstitutedanalogucsof substance P (SP), or of SI?, ,,, that function as substance P mcptor antagonists (Table II). Of these, j~At$, DT#:‘,L.~zu~‘JSP (spantide), IoArg’, ~Pro’, LIT@.‘, Lcu”~SP. (Fig. 1 and Table I) and loA&, oPheS,oTrP7.V,Leu1‘ISP arc three of the most potent”,?,‘. The second class includes analoguesof bombesin with o-amino acid substitutions for His12’7*z4. These peptides are more selective for bombesin receptorsthan the oamino actd-substitutedanalogues of substanceP, but are limited by low potency (Kit O.j_iO FM, Table II) and poor solubility. A third classof bombesin receptor antagonist includes various reduced peptirle bond analogues of bombesin. in thesepeptides the CONH group of the peptide bond is either changedto CH2NH (a vvbond replacement)‘~~‘or h to CH20

Peptide

EQmbesin(&Il

-12

[Leu”#~f0]Sn [Leu” vu-14pl (ophB~.cps':~~13-1418nbl,

4

p@uGin Arg Leu

GAP,,, U+t’W WsJmlnediil S Litorin [nPhe’TEl!l

3

5 GQ _t _

6

7

a

9

11

A.m fltu

_ _

_ _

__w--

-

Leu _

-w--s

-

-

^

_ly’_

ome

-

_

_

__--0

to

GR Tfp Ata Val Gty His _ _ _ -

_____

Thr _ _ -

_ _ _

13

14

HIS Leu _ _

12

Met _

NH* -

_

-

-

-

Leu Leu cpa

-

_

Phe Phe

DPhe -

-?#

--$I

neurcmedin C or GRflbz7:

(Table 1)22.The peptide [Leu”, y13-14)Bn (V, Ki 601~ Table II) was the first bombesin receptor antagonist described with sufficient potency to be generally usefults. A recent studylb reported a number of more potent members of this class of which the analogue ioPhe6,Phe”,yr13-14]Bn6_1,, (X Table Ii) is one of the most potent. The fourth cIass consists of desMet analo:ues of the C-terminus of GRP, ]desMeta7]GRP analogues or of bombesin [desMetr4]Bn anaIogues*Gzt (Table I and Fig. 1). These are currently the most potent and selective group r. antagonists. Of these the m ai potent antagonists are foPh&]&_ls ethyl ester. AC-CRPWa6 methyl ester and isobutyryl-[oAlaa]CRPae-26 methylamide (Ki l-10 nM) (Table III)ls2*.

action of bombesin; their affiniD-Amino acid-substituted ties for occupying the substance P aMloglle6 of SP receptor differed from those for The substance P receptor antagonist [DA~~,DR~~~DT~~~,~,L~~“]SP occupying born&sin receptors; no analogue increased the diswas the first member of this class sociation of boundU5lfryr’lBn; to be describedt4. Subsequent and neither substance P nor SP,_,, studies demonstrated that numercould inhibit the action of bomous o-amino acid analogues of beein. substance P or of SPctl, each of ‘Ihese anaiogues have also been which had substance P receptor mported to inhibit in oifro bornantagonist activity, also functioned besin-stimulated mitogenic skimuas a bomb&n receptor antaglation in 3T3 ceW, pq&ogen oni@. In one study it was conrelease from frog asophqeal cluded that six such analogues glandP and pancreatic enzyme (four of substance P and two of bombesinsecreHoxP2J. They have been SP& inhibited shown to block the in uino action stimulated enzyme secretion from of bombesin by some investipancreatic acini by functioning as gators%, but not by otherG. receptor antagonists since: each analogue inhibited binding of These peptides are limited by their lack of selectivity. Not only lasI~yr]Bn to pancreatic acini do they all act as substance P with the same relative affinities with whieh they inhibited the receptor antagonists (and in

2aa+3tt 46+10 5&l

94Of150 160+15 42k4

240*320 335f120 22Of42

13of7 55f5 7323

TiPS - /anuaty 1991 [Vol. 121

15

pwudppspt~? ? loPhe6, Cpa14. $ 13-141 Bns,, o bPhe6. Leui4, $13-141 Bnbrs A [Leu’j. J, 13-141 Bn 0 [Leti’, $ 26-271 GRP,_ [de&w] sn or [ckMan] GRP analogues ?? [oPhe6]Bn,,, ethyl ester ?? [oPhe6] Bn,,, NH, A [oPhe6] BnbtJ ethylamide + AC-GRP,, ethyl ester o-amino acid suktituted substance P analogues ? ? [DA@. DTrp’g, Leo”] SP n oPro’, ~Trp’‘. lo] SP,,, [DPIw’~ Bn antago&its J (oPt@, Leu”] Bn o [oPhe6,‘*] Bn

~100 E 8 ap 3 I

75

I! I E50 5 2

2

25

t

6 -10

-9

-9

-7

-6

-5

-4

Concentration (log M)

general have higher affinity for this receptor than for the bombesin mceptor) but some analogues also inhibit the action of vasopressin on 3T3 ceW or of cholecystokinin in pancreatic acinar cells=. They are also of limited potency since they are effective only in the micromolar range (Fig. 1). Several o-amino acid12 analogues of bombesin function as receptor antagonisW7*. The early studies of Broccardds demonstrated the essential nature of TrpS and Hisl2 of bombesin for biological activity. Subsequent studiesl”u investigating more than 100 different n-amino acid backbone-substituted analogues sug gested that substitutions in these positions might be particularly important in determining agonist or antagonist activity. It was found24 that compounds with substitutions in position 12, such as DPhe, D-p-chlurophenvialanine (D-Cpa) and ~Tyr - but not #he, o-pyridyldanine (D-Pal), ~Trp, ~Arg or D-knaphthylalanine (oNal) - acted as receptor antagonists, with [c#‘he*2,Leu*4]Bn having the highest affinity (III, KI 5-6pi, Table I). Additional amino acid replacements im-

proved affinity only threefold, with the (oPhe6J2]Bn analogue having the highest affinity (IV, Table II, and Fig. 1)“. These analogues have been shown to inhibit the CNS effect of bombesin on grooming behavior and bombesin-stimulated enzyme release from pancreatic acinisJ7J4. While these analogues are selective for bombesin receptors (although they do interact with substance P receptors in somes but not other’7 tissues) their efficacy is limited by both low solubility and potency and they have therefore not been generally useful for in vivo studies. Reduced peptide bond Bn analogues Reduced peptide bond analogues of gastrina and also of substance P, secretin and GRH have all been demonstrated to act as receptor antagonists. A similar strategy was therefore applied to bombesin15J6. When each peptide bond CONH group in the C-terminal octapeptide of bombesin’5 was replaced sequentially by a CHzNH group, four of the resulting pseudopeptides were agonists with relative affinities: (Leu’“]Bn = yllO-11>> 911-12 > 012-13 > I@-9. However, two of the pseudo-

peptides were antagonists, with VU-14 having a higher affinity than @-10. The [Leu*~,~l3-14]Bn analogue was the first bombesin antagonist to be described with affinity < 0.1~~15 and so was > 50-fold higher than that ot the [oPhe”]Bn or o-amino acid-substituted substance P analogues (Fig. 1). This peptide inhibits bombesin-stimulated pancreatic enzyme secretion in vitro and [SHjthymidine incorporation and autocrine growth in 3T3 cells and various SCLC cells1fJ6,31.In vivo it inhibits bombesin-stimulated gastric acid secretion, gastrin release and increased gastric motiIiw32. .fie mechanism by which this analogue functions as an antagonist is unknown. It has been pmposed33 that since bombesin has a p-turn between Vail0 and LeuD it may thus adopt an extensive hydrogen bonding configuration similar to that seen in somatostatin and LHRH analogue@, although this proposal is not universally accepted”. If such a p-turn existed, the increased rotational freedom introduced by insertion of the 1~1%14 bond would desh-oy the hydrogen bonding between the Leul3Leull CO grcup and the Ala9VallO NH group15.

TiPS - ]anuary 2991[Vol. 121

lb ~A~~~~~~.A~i~iesandpotenciesdanalaguesofdffMel"babasinanddedue(nGRP PMuemtkKdfd ml

auhbpis papride

&llm

IcrW

23m+230

296f26

226270

66+21

1600~310 96221

52OOi:1400 lOOk

>lOrKM 27k6

1600*75 29f16

12aOfl70 23*1

w-+3 6600+760

30-+11 6nY)t1600

2725 >I0000

0.7rto.3 >I0000

5+1 lfl 3kl lOtI

6fl 321 221 15-+2

22+1 10+2 Sfl l7_+1

2fl lkO.2 2.4+ 0.4 2.5-+0.1

Km)

JC,W 33210

216+30

11 h, Hz III loPbe 13Nb %k

61Ok230 24fll

IV @PheqBno_,~mi V [oPbe*~&ylami

7_+1 3400+560

I

Bn,-&H2

?'I [DPheqc+rl&waz~ VII [DPth+p?&Mhylester Vlllwhewe-,Jechurester IX N-Ac-Gf7P~&wfhyles@

4+1 2Jl 1.1k 0.3 4&l

3T3cew rc,lM

~CS&M

3.3fO.l 6099+960 2fl 1.1f0.2 2fl 2fl

m-fromprmxesticaeMa_ !c,Whehecarcentrabkrnusinglmll-maximalinhibitbndbom~ truqwaM bllo 3T3 c&s'"'? ~valuescdaJalsd~md~~~~~d~d~~‘~~~~~d theceHsystems.611, bombssin: GRP,@rin-releasingpeptide;kacetyl.

A recent study’6 has identified short chain pseudopeptide bombesin receptor antagonists that are more potent than [Leu”,+ 14]Bn, may have fewer proteolytic degradation sites and can be more (Table II). Bns-ll easily synthesized or CRl’&, are the minimal (and low affinity) C-terminal fragments with biological activityzneJ5whereas the nonapeptide Bn,,-l~ has almost equal affinity with native bombesirW5. The nonapeptide pseudopeptide of bombesin (VI, Table II) and the pseudopeptide of the decapeptide GRPIs-?, (VII, Table II) are both bombesin receptor antagonists although of much lower affinity than [Leu1J,q1314jBn (V, Table II); however, the $g-9 pseudopeptide of the nonapeptide litorin (Table I; VIII, Table II) shows a higher affinity in this system. This peptide differs fmm [+13-14]Bnbll in having a p-Glu at position 6 of bombesin and a Phe at position 13 (Table I). Because the insertion of oPhe into position 6 of the [oPhe**]Bn antagonist increased affinity21 a similar insertion was made in the bomb&n nonapeptide pseudopeptide ilx, Table II), and a 50fold increase in affinity was

achieved. The insertion of Phe, DPhe or Cpa into position 14 (Table I, Fig. 1; XI, Table II), increased potency an additional twofold in 3T3 cells, resulting in antagonists with affinity in the 3-10 KMrange’b*36(Fig. 1). Additional studies with both pseudopeptides and IdesMeWBn analogues*bJs demonstrated that most substitutions in position 6 of bombesin resulted in minimal loss of antagonist affinity (XI, XII, Table II). In tbg pseudopeptide, the replacement of the C-terminal NH2 with OH or the further shortening of the chain length to Bn,_,, markedly reduced affinity (XIII, XIV, Table II). Recent studies~ suggest that reduced peptide bond analogues with CONH substituted by CH20 may result in analogues with even higher affinity. One such analogue, AC-& CHfl25-26]GRP~~, had a K! :Lf 30 nM for 3T3 cells and removal of the terminal carbamoyl group reduced the K, value to 10 n@. IDesMeP41Bn/idesA4e~GRP anaiogueantagonists Previous studies with cholecystokinin or gastrin*sJ’ demonstrated that C-terminal desPhe

amidated analogues were either potent antagonists or partial agonists. A similar strategy has been applied to bombesin or GRP by a investigatorsls-P number of (Table I). The analogues AcAc-CRP~H~ GW&‘JH2, and Bnl_,sNH2 (I, Table III) were all shown to function as weak bombesin receptor antagonists in 3T3cells orin pancreaticachW*. As with the pseudopeptides, shortening of the chain lengthof bombesin to the nonapeptide (II, Table III) significantly reduced potency, but insertion of a [Ph&] into the desMet” nonapeptide increased potency > 20-fold (III, Table III)“j**s. The formation of alkylamides (IV, V, Table I@ also increased potency and the combination of a DPhd and a C-terminal alkylamide resulted in a bombesin analogue with a Ki in the nanomolar range (IV, Table III)” (Pig. 1). Similar mults were obtained with two GRP analogues: Ac[Dtiaz4]GRPmethylamide or AC-GRP,, ethylamide have affinities from ZO-5OnM for 3T3 cells2021. These results*s~~ demonstrate that the C-terminal amino acid of bombesin-related peptides is im-

TiPS - Janna y 2991 /Vol. 121

17

TABLEV.AffinMsandpoteMesofbcmbesinreceptwagonistsandantagoniis R&pamrr EGdW -. zP@m GRhbn newanedinB

-ai+¶dwi-,mester

a.2?I0.1 0.3* 0.1 0.4f 0.2 651

Rilt-wN@rnnmraw K(mc)

ECso(W

4+1 15+3 20+12 350225

5+2 39k19 113f3G 4*1

2fl 30+4 39+3 0.30A 0.03

GdW llOmfm0 35omf12OaI Nlat3Optd Nlatlp Nlatlpu NlatO.luu

&1:,13000~109 mO+1ooo 4409Of2lm 59000+4clOo 52000*5000 50090*9009

Go(W h(m) 39100*9990 13m9f29w NT@riialeigolw) 70000f4000 210259 430+60 lo+1 4Of5 1522 19k5 2 f 0.4 5+1

portant for initiation of biological mal fragment that both interacts response, but is not essential for with the bombesin receptor and determining receptor affinity. The also elicits biological activity**J5. mechanism by which the desIn some@ but not aI13cell systems Met” bombesin analogues functhe C-terminal nonapeptide was tion as antagonists could be the the minimal fragment to be equisame as that 0riginaIly proposed potent with bombesin or GRP. for the pseudopeptides i.e. the Studies wi’h analogues of pseudocarboximide at position 14 in peptidebombesin,GRP, [desMetia]bomb&n is essential for exbombesin and [desMflGRP pression of agonist activ+. have ail led to the conclusion that Moreover, the increase in antag the C-terminal methionine is onist affinity resulting from the essential in determining biologiaddition of alkyl groups at poscal activity, but not in determinition 13 may be due to the electmn- ing affinity for the receptori~~“‘J9~2’. However, recent studies demonreleasing properties of the alkyl strate that Met14 per se is not substitutionsis, perhaps by enessential for biological activity, hancing hydrogen bonding in bombesin/GRP receptor intersince bombesin ana!ogues with Phe, Leu, or Nle substitutrd at actions. Recent studies support position 14 all have agonist acthis conclusion; formation of tivity*“. Indeed, the determinants desMet” bombesin or desMetn of biological activity are more GRP an-es with other eIectroncomplicated than was previously releasing ~;roups such as hydrathought’9+ zides (VI, Table III) or esters (VIIA number of pseudopeptides IX, Table III) are very potent bomand C-terminal desPhe amidated besin receptor antagonists1921. analogues of cholecystokinin or Various ester analogues such as ester19, gastrin’9*Jr have partial agonist ethyl [uphe61%-is activity in some, but not all, AC-GRP&% ethyl estera’ and triacetyl-[oAlaz4]GRP20-2, species. ‘This was not found to be methyl the case in a series of more than methyl este9 have been shown to 150 bombesin analogues. Howbe potent bombesin antagonists ever, [Leutd, yr13_14]Bn,which is a both in vitro and in vivo, as asrelatively potent bombesin antagsessed by inhibition of bombesinonist in 313 cells or guinea-pig or GRP-stimtdated amylase release pancreatic aciniis, shows weak or gastrin reIeasei9JaJs. The anapartial agonist activity in frog es+ logue isobutyryl-[nAlaz4]GRPzo_w phageal peptic cells39, suggesting methylamide (Table I) may have that some classes of purported an additional advantage of a bombesin receptor antagonist slightly longer duration of action may have partial agonist activity in vivo. in certain species. In almost all cases, the various classes of bomDeterminanb ofagonist/ besin receptor antagonist were antagonIst aftlvity initially tested either in 3T3 cells The C-terminal heptapeptide of or in guinea-pig pancreatic acini. bombesin or of GRP is the mini-

K,(W

In recent studiesi the abilities of a number of bombesin analogues to function as bombesin receptor antagonists in guinea-pig pancreatic acini were compared to their interaction with bombesin receptors in 3T3 cells and in rat pancreatic acini. Similar studies examining the ability of cholecystokinin-related peptides to function as chokzcystokinin receptor antagonists showed that structural requirements for biological activity in rat pancreas were much less stringent than in the guineapig pancreas. When 32 bombesin pseudopeptides, IdesMetr41Bn and (desMemGRP analogues were assessed in this way there was a very close corrclat:on in the peptide structural determinants of biological activity for 3T3 cells and guinea-pig pancreas, but not for rat pancreatic acini (see Table IV). Of the eight representative analogues in Table IV, two had partial agonist activity in guinea-pig oancreas or 3T3 cells, whereas six were potent antagonists. By contrast, in rat pancreas four analogues demonstrated agonist activities and four were antagonists. Furthermore, a number of analogues (such as VII, VIII, Table IV) that were partial agonists in 3T3 cells and guinea-pig pancreas were full agonists in rat pancreas, and some analogues (such as I, VI, Table IV) that had antagonist activity in these two cell systems were partial agonists in the rat panaeasr9. These results suggest that rat pancreatic bombesin rec~ptom have less stringent peptide sbuctural requirements for activation as was observed (above) with re??

TiPS - Janus y 1991

IS

/Vol.

I21

a high affinity for bombesin, GRP and GRPIs_27,and a 20-fold lower affinity for neuromedin B (Fig. 2, Table V). Bombesin and litorin have a relatively (Table V) high affinity for each of these different bombesin receptor subtypes. In terms of its selectivity for these different naturally occurring peptides, pancreatic acini bombesin receptors resemble those of 3T3 cells, rat gastric, uterine and colonic muscle ships, isolated guinea pig gastric smooth muscle cells, various areas of the CNS, SCLC cells and pituitary cell

80 60 40 20

line3,12,42A7.

20 t 100 80

ceytors for cholecystokininrelated peptides. The differences observed in responses in these three different cell systems led to the development of analogues that are antagonists in vitro in each systemr9,36. When desMet**Bn analogues were modified by increasing the chain length of the alkylamide (up to propylamide, Table IV), the potency in each cell system was markedly increased. However, when the analogues contained an alkyl group longer than propylamide partial agonist activity was observed in 3T3 cells or guinea-pig pancreas; analogues longer than ethylamide had partial agonist activity in rat pancreas (V, Table IV)19.All ester analogues as well as the hydrazide analogue were potent antagonists in each cell system (2-10 mu) (VI-IX, Table i11)19.Almost all of the pseudopeptides had weak bombesin receptor-mediated partial agonist activity in rat pancreas19*. However insertion of either Cpard or

DPherJ (analogues II, III, Table IV) resulted in potent antagonists in each cell system%. These results suggest that the [desMett*]Bn or (desMeP]GRP ester, hydrazide or ethylamide analogues and the pseudopeptide analogues with Cpar* or uPher (II, III, Table IV) may be the most universal antagonists. Differentiation of Bn receptor subtypes by a&ago&b Preliminary evidence in gastrointestinal tissues and in the CNS suggests the existence of two subtypes of bombesin recepto?. A bombesin receptor with high affinity for neuromedin B and a significantly lower affinity for GRP or GRPis_zr was identified in rat esophageal muscularis mucosa42 (‘Table V, Fig. 2), in isolated gastric smooth muscle celW and in the CNS4s.a. By contrast, in pancreatic acini.2 the bombesin receptor mediating stimulation of enzyme release has

A number of bombesin receptor antagonists also have markedly different affinities for these proposed subtypes of bomb&n receptorWr (Fig. 2, Table V). These results suggest that, at present, some of the new potent bombesin receptor antagonists can distinguish the ability of bombesin to mediated changes in biological activity by two different receptor subtypes. At present potent recaptor antagonists exist only for the bombesin receptor subtype that has a high affinity for GRP such as occurs in pancreas, 3T3 cells, SCLC cells, some areas of the CNS, gastric smooth muscle cells and pituitary cells. No potent antagonist has been identified for the bombesin receptor subtype that has high affinity for neuromedin B. Acknowledgements This reseaxh was partially supported by NIH Grant CA45153. References 1 Tache, Y., Mekhioni,

P. and Negri, L.. Bonrbesin-lilrr Peptides in He&k and Cl&% IAnn. NY Acod. Sri. Vol. S47). pp. 1-540 2 Ghatei. M. A. et PI. (19s2) I. Clin. Endocrittol. &tab. 54,980-W 3 Jensen, R. T. rt of. (1988) Ann. NY Acad. Sri. 547, US-149 4 Enpamer, V. and Mekhioti, P. (1973) Pore Appt. Chcm. 35,4&X-493 5 Ruff, M.. Schiffman, E.. Terranova. V. and Pert, C. P. (1965) C/in. In~~~rr~ol. Imma~topafhol. 37.387-3% 6 Cowan, A. (1988) Am NY Acud. Sri. 547,204-209 * 7 Brown, M. R., Carver, K. and Fisher. L. H. (1988) AM NY Arad. Sci. 547, 174-182 8 Merali, 2. ct al. (19.38) Syorlpse 2. UC287 9 Cuttitta, F. ct RI. (1985) Nalwc 316. 823-926 10 Wllley, J. C., Lechner, 1. F. and Harris, C. C. (1984) Exp. Cc// Ra. 153, 245-248

eds (1986)

TiPS -January

1991 /Vol. 721

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Rorenguet, E. (19s8) Proc. Natl Acad. Sci. USA 85,1859-1863 12 Rozengurt, E. (19BB) Ann. NY Acod. Sri. 547.277-292 13 Mahmoud. S., Palaszynski. E., Fiskum, C., Coy, D. H. and Moody, T. W. (1989) Life Sci. 44.367-373 I4 Jmsen, R. T., Jones, S. W., Folken, K. and Gardner, J. D. (1984) Nature 309, 61-63 15 Coy, D. H. et al. (%%I) /. Biol. Chem. 263, 50%5060 16 Coy, D. H. et al. (19B9)/. Biol.Chem.264, 14691-14697 17 Heinz-Erian, P. ef at. (1987) Am. /. Physiol.252, G439-G442 18 Wang, L-H. et nl. (1990) Biochemisfry29, 616-622 19 Wang, L-H. et al. (19%) /. Biol. C/rem. 265,15695-15703 20 Gamble, R. et nl. (19B9) Life Sri. 45, 1521-1527 21 Heimbrook. D. C. et nl. (1989) /. Biol. Chcm. 264,1125&11262 22 Saari, W. S., Heimbruok, D. C., Friedman. A., Fischer, T. W. and Oliff, A. (19@) Biocbmt. Biophys. Res. Commun.165, 114-117 23 Jmsen, R. T. et al. (1988) Am. /. Plfysiol. 254, GBB3-GB90

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33 Rivier, 1. E. and Brown, h4. R. (1988) Biochemistty 17, 1766-1771 34 Eme, D. and Schwyzer, R. (1987) Biochemistry26,63X-6319 35 Heimbrook, D. C. et PI. (1988) !. Biol. Chem.263,70X-7019 3& Coy, D. H., Wang, L-H., Jiang, N-Y. and

Dierential modulationof tissue functionand therapeutic potentialof selectiveinhibitors of cyclic nucleotide phosphodiesteraseisoenzymes C. David Nicholson, R. A. John Chaliiss and Mohammed Shahid Since the discovertt of cyclic nucleotide phosphodiesterase 30 yearsago, there have been major advances in our knowledge of this group of isoenzymes. Five families, each composed of several isoforms and having differing tissue distributions, have beendescribed.David Nicholson and colleaguescompare the tissue distribution of phosphodiesteruse isoenzymes and discuss the differelztial effects of inhibition of particular isoenzymes, with differin subcellular localization, on tissue function. They also review the potential use of &enzyme selective phosphodiesterase inhibitors in a range of clinical disorders such ns heart fnihrre, asthmn, depression and dementia. Cyclic nucleotide phosphodiesterases (EC 3.1.4.17) play a key role in the metabolism of CAMP and cGMP. Many agents modulate tissue function via stimulation C. D. Nicholsotris Head of Departaerrt of Phnrmncofology and M. Shnhid is R Phnnnaco/o&t in the Heart Foifrre Cro~cp, Uqo~toti Laboratories Ltd. Newbow ML1 SSH, UK. R. A. 1. ChRiss is Wrllromr Lwtttrer b! BiorhcmicalPhanaacol~y in fhr Departmetit of Phrrmarof~y nttd lhempertirs. Usiurrsity of Leicester.PO Box 131, f&ester LET YHN. UK.

of adenylyl or guunylyl cyclase activity and hence via the elevation of cellular levels of CAMPand cCMP. Phosphodiesteraseactivity moderatesthe effectof theseagenk by increasing the rate of breakdown of these cyclic nudeotides. The existenceof families of phosphodiesterase isoenzymes is well recognized and isoenzyme selective inhibitors have been identified’. A recent TiPS review* summarized our knowledge of the

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structure, regulation and some aspectsof the localizationof phosphodiesteraseisoenzymes; it also proposed a nomenclature for the identified isoenzyme families. This review further compares the tissue distribution of phosphodiesterase isoenzymes, describes how selective inhibitors can differentially modulate both cellular cyclic nucleotide levels and tissue function and discussesthe clinical potential of phosphodiesterase isoenzyme selective inhibitors. Comparativetissuedistribution The nomenclature used for the phosphodiestenseisoenzymes, their most salient regulatory characteristicsandsubstratespecificities, as well as the pharmacological profile of representative isoenzyme selective inhibitors, is summarized in Table I. A chro-

matographic elution profile illustrating the phosphodiesterase isoenymes in cardiac mu& is shown in Fig. 1. Although early studies were performed with lowresolution techniques, sufficient data are now available to illustrate clear tissue differences in phosphodiesterase isoenzyme distri-

bution, For example, PDE I, PDE III and PDE IV represent more than 90% of the total CAMP phosphodiesteraseactivity in brain, platelet and kidney cells,resputiively’,2. Table I! summarizes results from recentstudiesthat included appro-