Antimycotic sterol biosynthesis inhibitors

233

TIPS - J u n e 1986 amines (Blaschko, H. and Muscholl, E., eds), Vol. 33, pp. 845-899,Springer 9 Rajfer, S. I., Anton, A. H., Rossen, J. D. and Goidberg,L. I. (1984)N. Engl.J. Med. 310, 1357-1362 10 Goldberg,L I. (1972)Pharmacol. Rev. 24, 1-29 11 Chidsey,C. A., Braunwald,E., Morrow, A. G. and Mason,D. T. (1963)N. Engl. J. Med. 269, 653--658 12 Kramer, R. S., Mason, D.T. and

Braunwald, E. (1968) Circulation 38, 629-634 13 Petch,M. C. and Nayler,W. G. (1979)Br. Heart J. 41, 340-344 14 Goldberg, L. I. and Rajfer, S. [. (1985) Circulation 72, 245--248 15 Borow, K. M., Lang, R., Neumann, A., Carroll, I.D. and Rajfer, S.I. (1985) Circulation 72, III-304 16 Borow, K. M., Come, P. C., Neumann, A., Baim, D.S., Braunwald, E. and

Antimycotic sterol biosynthesis inhibitors D. Berg, K.-H. Bechel, M. Plempel and E. Regel The last decade or so has seen the development and increased use of the antifungal azoles. D. Berg and colleagues describe the pharmacology and medicinal chemistry of these compounds which has allowed research in this field to move towards the developmen~t of analogues with reduced toxicity suitable for oral and parenteral administration.

The n u m b e r of mycological infections in h u m a n beings and animals is continuously increasing. The most important h u m a n mycoses and their causative organisms are listed in Table I according to their statistical importance. Since the discovery of the first antifungal azole compounds clotrimazole and miconazole in 1967 and their commercial availability, a n u m b e r of antimycotic azoles have been developed w i t h i n the last 12 years. The therapeutic situation in dermatomycoses as well as fungal infections of the vagina has consequently changed dramatically. The azoles s h o w - beside a broad antimycotic activity - an exceptional skin and mucous m e m b r a n e compatibility. Their enormous antimycotic activity allowed a drastic reduction of duration for treatment of mycoses, e.g. for vaginal mycoses from 3 weeks to 1-3 days as well as a reduction of n u m b e r s of applications from 2-4 times to once daily. The resulting increase in patient compliance reduces the n u m b e r of relapses. Experimentally m a n y azole derivatives exert antimycotic activity after topical and oral, as well as parenteral application. After systemic administration they are potentially hepatotoxic and may exert - according to their specific The authors are research workersat BAYERAG, 5090 Leverkusen-Bayerwerk, FRG.

mode of action - effects on h u m a n testosterone and corticosteroid synthesis. Teratogenicity and embryotoxicity are sometimes observed in animal experiments after systemic azole administration. In spite of these potentially serious side-effects it will be possible to select those from the large n u m b e r of antimycotic azoles which possess a decreased potential for side-effects, together with an additional feasibility for reduction of duration of treatment. This makes oral and parenteral administration possible in principle. C h e m i c a l nature of azole antimycotics

The most important imidazoles and 1,2,4-triazoles which are in clinical use or u n d e r development are listed in Fig. 1. All possess a heterocyclic aromatic substituent with an N-atom in the 3-position as shown in the general structure (Fig. 2). The fungistatic nature of azoles involves characteristic morphological changes such as thickening of cell walls, increased branching, incomplete septa formation, considerable vacuolization, as well as accumulation of lipid bodies. The primary mode of action appears to be disorganization of the fungal plasma m e m b r a n e 1-3. Membrane function is obviously disturbed after application of low concentrations of azoles, leading to

Grossman, W. (1985) Am. J. Cardiol. 55, 1204--1209 17 Colucci, W. S., Alexander, R.W., Williams, G.H., Rude, R.E., Homan, B. L.,Konstam,M. A.,Wynne,J.,Mudge, G.H., Jr. and Braunwald, E. (1981) N. Engl. J. Med. 305, 185-190 18 Kenakin,T. P. and Ferris, R. M. (1983) ]. Cardiovasc. Pharmacol. 5, 90-97 19 Daly,P. A., Curran,D. and Chatteljee,K. (1985) Circulation 72, III-406 changes in membrane permeability, accompanied by alterations of specific activities of membranelocalized enzymes4, finally resulting in i n h i b i t i o n of growth or death of the fungal cell. These morphological changes are due to azoleinduced interference with synthesis of ergosterol, an important component of fungal membranes which enhances the physical membrane stability by complexing with phospholipids s. Ergosterol is a quasi-planar molecule which is able to stabilize phospholipid phases 6. Lack of ergosterol, as well as accumulation of n o n p l a n a r sterol precursors, consequently leads to disruption of fungal membranes. After addition of azoles to suspension cultures of h u m a n pathogens 24-methylenedihydrolanosterol (Fig. 3), a nonplanar precursor of ergosterol, accumulates, indicating that the subsequent step in sterol synthesis is inhibited, i.e. the oxidative demethylation at C-14 (Ref. 7). The enzyme complex responsible for the oxidative removal involves a cytochrome P-450 system, and a direct interaction between the Feporphyrine complex (the prosthetic group of the P-450 enzyme) and different azoles has been proven 8. Direct interactions between the ferric ion and inhibitors have been studied by difference spectroscopy and shown to represent a type II i n h i b i t i o n8. Side-effects

This knowledge is of fundamental interest w h e n considering the severe toxicological sideeffects which have been observed during azole studies, e.g. inhibition and/or induction of liver cytochrome P-450s and a possible teratogenicity, which is strongly structure dependent. Our present knowledge allows a speculative interpretation of these findings9. The liver cytochrome P-450 isozymes in general are responsible for metabolism of xenobiotics and thus have to be able to accept a

~) 1986, Elsevier Science Publishers B.V., Amsterdam

0165 6147/86/$02.00

TIPS -June 1986

234

Compound

Form~

~

F W

fon'o

cl

cl

ci ~

Miconama~oke

Cl

1

H---O---CH2~

H2

~ruizole

CÀ

H---O---CH2

i

Topical LV.

Topical

C1

To~l

Tioconuole

H2

BAY 1 9138

C1

H-'-CH---C ( CH3 ) 3

,H2

BAYn 7133 Vibunuole

Qo~ma=o~

~ - - ~

,oH2

Topical

C1

S~JA70 Bifonuole

~

H

~

Cl

Ond

---CH2--S--fCH2)3~H 3 ~H2

Topical

Cl

cI

CH2 -

/~.

N

Oxl]

TOpiCal

IIx~co~L~le

ru~x~i~ole CH2-O~N~_

/ N-COCH 3

C4H9-s C1

IIK)COlldIZO~

C1 __~:/ )H----O----CH2

Topical

C1

Topi~

O~dcom:m~e

,H2

Cl Texcomlzo]e

CX ~ , ~ - - - - - C H

2--N/~'~

Topical

Fh~onazole

Oral CH2

Fig. 1. Structural formulas of commercially available and developmental antimycotJc azoles.

broad variety of different chemicals as substrates; thus substrate specificity of necessity appears to be rather low. The reverse is true for the biosynthetic P-450s involved in estrogen synthesis, e.g. the steroid16-, steroid-17-, and steroid-19hydroxylases 1°. Azole antimycotics interfere with these e n z y m e s directly 11. Since reduced estrogen synthesis is a possible conse-

quence of azole-induced teratogenicity 12, it could be speculated that inhibition of the rate-limiting aromatase could be crucial for the teratogenicity of a given azole test compound. On the other hand, as 24-methylenedihydrolanosterol demethylation is also a biosynthetic monooxygenase reaction and therefore requires a substrate-specific cyto-

chrome P-450. As both the nature of the substrates as well as positions of methyl groups to be oxidised vary for the different P-450 e n z y m e system, the possibility of selective inhibition also exists. In general, the present goal in azole chemistry is to m a x i m i z e efficacy against the fungal e n z y m e while m i n i m i z i n g activity towards liver P-450s and aromatase.

235

TIPS -June 1986

R1

I

T,--c--~

I ~N~J L = lipophilic (normally aromatic) substituent R~, I~ = H, alkyl, aryl, etc. (at

X~

least one lipophilic substitution)

N - Nitrogen in distance 3 of the bridge atom, of a heteroaromatic zing, e.g.

Oral administration preseuts more problems since compounds must be absorbed and certain plasma levels must be achieved for systemic distribution. Such azoles have to be optimized with respect to liver and placental enzymes and only a few compounds fulfil these conditions, e.g. ketoconazole, oriconazole, vibunazole, and fluoconazole. Even in these cases, liver enzymes should be controlled during treatment, and administration during pregnancy is to be avoided 12. Nevertheless the prevalence of severe, even lifethreatening, organ mycoses is more than sufficient ethical justification for the development of such compounds. Resulting from observations

Fig. 4 shows the C-14 demethylation reaction on 24-methylenedihydrolanosterol where, after hydroxylation, the C-14 carboxylic acid is eventually formed. This is not decarboxylated directly, but formic acid splits off by taking a proton from C-15 thus leading to a A14-double bond which is then reduced in a NADPH/H+-depen dent reaction to yield the endproduct. The morpholines are inhibitors of such N A D P H / H +dependent reactions TM (Fig. 5). Tridemorph and fenpropimorph are of great significance as agricultural fungicides against powdery mildew in cereals. Ro-144766/002 exhibits high antimycotic activity, particularly against yeasts. These agents are also useful tools in

TABLE I. Most important and frequent human mycoses

-

imidazole 1,?.,4-triazole 3-pyridine 5-pyrimidine

Mycosis

Causative organisms

Dermatophytosis (ringworm)

Trichophyton species Microsporon species Epidermophyton floccosum

Vaginal mycoses

Candida albicans Torulopsis glabrata other Candida species Candida albicans other Candida species chromomycetes, moulds

Fig. 2. Genera/structure of azoles.

Various fungal infections of the skin and mucous membranes

Mode of administration The necessary degree of selectivity towards these enzymes, however, depends on the mode of administration of the active ingredient. Some infections can be treated locally, e.g. topical dermatophytoses and most vaginal infections. On the other hand internal organ mycoses have to be treated by oral administration of a systemic compound. Accordingly, these two forms of administration put fundamentally different requirements on the properties of a candidate drug. If an azole is to be administered topically, selectivity for the fungal enzyme need not necessarily be very pronounced provided - and this is most important - the compound is not absorbed to a great extent. An example of this kind of drug is bifonazole, which is applied topically. Absorption through the skin is very limited 13 but bifonazole remains for a long time within the fungal cell; even sub-lethal concentrations still lead to a partial inhibition of sterol synthesis, thus nullifying pathogenicity 14 ' 15 . Compounds with this profile are not only ideal for topical administration but also allow short-term therapy due to their prolonged effects on the pathogen.

Systemic mycoses: (1) by opportunistic fungi

(2) by primary pathogenic biphasic fungi

with experimental products, the following goal seems to be consistent for such a class of therapeutics: azoles should be rigorously minimized with respect to non-specific inhibition of non-target cytochrome P-450 enzymes. Furthermore, short-term or ultra-shortterm therapy has to be the aim, especially for oral administration. This has already been realized in topical treatment, e.g. single-application clotrimazole treatment. If this could be achieved for oral azole drugs it would help a great deal in optimizing patient compatibility. Modeofaction Research on the mode of action of azoles has focused on studies of sterol biosynthesis, particularly on inhibition of mono-oxygenases.

Candida albicans other Candida species Aspergil/us species Cryptococcus neoforrnans Zygomycetes a.o. Histoplasma, Coccidioides immitis Paracoccidioides brasiliensis B/astomycesspecies Sporothrix schenckii

sterol synthesis research. Another group of compounds interfering with fungal sterol synthesis has been discovered recently. The allylamines, represented to date by just two compounds, naftifine and terbinafine (Fig. 6), are most interesting in that they

Fig. 3. Structural formula of 24methylenedihydrolanosterol.

236

TIPS - June 1986 TABLE II. Azole antimycotics: spectrum of antimicrobial activity and MIC-values MIC-values in Lugm1-1 (Rafs 17-20) Fungal and bacterial species

Dermatophytes

l ~ cytochromeP-4S0

HO~/~-.J Icytochrome P-4~- H~O

( Trichophyton, Microsporon, Epidermophyton) Candida spp. Torulopsisglabrata Aspergillus spp. (A. fumigans, A. niger, A. nidulans) Chromomycetes Biphasic fungi

(Histoplasma, Coccidioides, Paracoccidioides, Blastomyces)

HO~

P-4,qO

~"

~HCOOH

2"~t--- NADPH/H*

Fig. 4. Proposed reaction sequence for C- 14 demethylation.

inhibit a different step in ergosterol synthesis, the epoxidation of squalene, which is again a monooxygenase reaction (Fig. 7). This is a prerequisite for cyclization to lanosterol. In principle this biosynthetic step is not pathogen-specific as epoxidation of squalene is also involved in cholesterol synthesis in mammals. A pronounced toxicity of these compounds would therefore be expected, and it is surprising that remarkable differences of this target clearly exist between mammals and fungi since terbinafine has been shown to be orally compatible. Antimycotic imidazole and triazole derivatives have a very wide spectrum of activity embracing practically all fungi pathogenic to humans and animals. Only zygomycetes - M u c o r a n d Absidia species - which can be pathogenic in humans under certain metabolic conditions such as severe, uncontrolled diabetes, are primarily resistant. The spectrum and intensity of activity of this class of antimycotics

are illustrated in Table II for three well-known topical imidazoles 17-2°.In the case of azoles, the results of MIC (minimum inhibitory concentration) determinations are highly dependent upon the test methodology used. Apart from the inoculum size, the composition of the nutrient medium and the physiological condition of the fungal cells are highly critical test parameters. For instance, the parasitic mycelial forms of C. albicans are 10-20 times more sensitive to azoles than saprophytic budding cells - simply because long fungal hyphae require more ergosterol for the construction of the cytoplasmic membranes than round budding cells. MIC determinations with azoles must therefore be adjusted to the physicochemical and biological properties of the substance in order to avoid false-negative data. As test medium we recommend Kimmig or DST standard media. The optimal inoculum size

C13H27--N

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Clotdmazole

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0.25-2

0.25-1

0.25-4

1-4 0.25 0.5-1

0.25-1 1-16 0.25-1

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1-4

1-2

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0.25-2

0.5-2 0.25-1

1-4 0.5-16

Corynebacteria Gram + cocci (without enterococci)

/•/CH3

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2-16 2-16

is 103-104 CFU m1-1 (Ref. 21). In addition to their antifungal activity, the azoles also display invitro activity against Grampositive cocci (with the exception of enterococci), and corynebacteria. Gram-negative bacteria are either of low susceptibility or are primarily resistant 19m. The azoles have a concentration-dependent variation in type of action, ranging from partial fungistasis at subinhibitory concentrations in the nanogram region to an impressive and complete fungistatic (MICzoo%)and finally to fungicidal action at 4--10 times the MIC values. Most azoles act fungistatically in the therapeutic relevant concentration range. However, some azoles, e.g. bifonazole, block two sequential reaction steps in ergosterol biosynthesis, and have a consequent fungicidal effect against filamentous fungal elements even at concentrations reached at the site of infection during therapeutic use 21.

~H3 tC4H9 ~

-

~

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~ CH3

\

Tridemorph

Fenpropimorph

c,. C2H5-

0.25-16

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\

Ro- 14-4766/002 Fig. 5. Morpholine ergosterol-biosynthesis-inhibitors.

/~ CH3

CH3 O /~'CH3

TIPS

-

237

June 1986

Naftifine

Terbinafine

Fig. 6. Structural formulas of naftifine and terbinafine.

Resistance The azole class of drugs has to a large extent escaped resistance. The primary resistance level within susceptible fungal species is a m a x i m u m of 1-2%. Secondary resistance development occurs, if at all, according to a multi-step scheme and to date can be disregarded, both epidemiologically and therapeutically. Nevertheless, the sporadic observations of primary or secondary resistance should still be carefully analysed in the future. Such p h e n o m e n a recorded to date have been mainly due to errors in in-vitro testing; reports on resistance cannot be reproduced w h e n test methodology is standardized 21'22.

Azoleuptake Even after contact times as short as 15-25 min there is a m a x i m u m intracellular uptake of an azole, which then remains w i t h i n the fungus for more than 120 hours. This leads to a prolonged reduction in the ergosterol biosynthesis and, as a consequence, to a marked loss of virulence in vivo of azolecontaminated fungal o r g a n i s m s . Cells of Candida albicans subjected to a short (15 min) in-vitro contact with

s u b i n h i b i t o r y concentrations of clotrimazole or bifonazole, for example, were found to be no longer infectious in a s u b s e q u e n t animal experiment 23. On average, only 1-3% of

% Ergosterol 100 ~

2 cm = 10% 2cra = 10h

C]otri_mazole

80 70 60

~----""~ontrol Bifonazole

lo

1'o

2'0

3'o

I

40

50 60 70 80 90

I

I

100

110

J

120

Time (hours) Fig. 8. Partial inhibition of sterol synthesis in Trichophyton mentagrophytes after application of O.1 pg m1-1 clotrimazole and bifonazole and transfer to azole-free medium.

topically applied azoles is absorbed dermally. Accordingly, serum concentrations of less than 1-5 ng m1-1 have been found in h u m a n s u p o n topical administration of 1% azole formulations to skin areas of ca. 200 cm 2.

\/

Squalene

For intravaginal administration absorption, rates of up to 15% can be found for i n d i v i d u a l azole drugs, but generally, absorption rates are on average less than 3%. Upon oral administration in h u m a n s and experimental animals, more than 60% of m a n y azoles are intestinally absorbed. Peak serum concentrations occur 2-4 h after administration. All the azole derivatives reported to date that have been examined u n d e r conditions of oral administration have been degraded in a first-pass reaction b y microsomal liver enzymes to microbiologically inactive metabolites and more than 90% of the administered dose eliminated via the faeces. During metabolic breakdown they can cause a rapid liver enzyme induction and serum levels fall continuously as a function of

Squalene-2,3-epoxid

Fig. 7. Inhibition of squa/ene-epoxidation by ally/amines.

the duration of treatment. Species differences are common for this type of reaction. With two exceptions, miconazole and ketoconazole, the azole derivatives currently available commercially are only suitable for topical and/or intravaginal use. Miconazole, which has proved of great value as a topical antimycotic, is only a drug of second choice as an i.v. formulation owing to pharmacokinetic drawbacks and problems with patient compatibility24. Ketoconazole is active as an oral antimycotic but limited in its indications b y side-effects on the liver and on testosterone/cortisol synthesis 25-27. As topical and intravaginal antimycotics the azoles are the drugs of choice for all dermatomycoses and mycotic infections of the vagina.

238

References 1 Beggs, W. H. and Sarosi, G. A, (1981) Life

Sci. 28, 111-118 2 Borgers, M. (1980) Rev. Infect. Dis. 2, 520-534 3 Medoff, G. and Brajtbury,J. (1983) Ann. Rev. Pharmacol. Toxicol. 23, 303-330 4 Berg, D. (1986)ACS-SymposiumSeries, 304 5 Dahl, C. E., Dahl, J.S. and Block, K. (1980) Biochem. Biophys. Res. Commun. 92, 221 6 Rottem, S., Cirillo, V. C., de Kruyff, B., Shimibky, M. and Razin, S. (1973) Biochim. Biophys. Acta. 323, 509 7 van den Bossche, H., Willemsens, G., CoUs,W., Lanwers,W. F. J. and le Jeure, L. (1978) Chem.-Biol. Interact. 21, 59-78 8 Wiggins, T. E. and Baldwin, B. C. (1984) Pestic. Sci. 15, 206-209 9 Kellis, J. T., Sheets, J.J. and Vickery, C. E. (1984) J. Steroid Biochem. 20, 671-

TIPS - June 1986 676 10 Kellis, J. T. and Vickery, L.E. (1984) Science 225, 1032-1034 11 Loose, D. S., Kan, P.B., Hist, M.B., Marcus, R.A. and Feldman, D. (1983) J. Clin. Invest. 71, 1495-1499 12 Schiirmeyer, Th. and Nieschlag, E. (1984) Acta Endocronol. 105, 275-280 13 Liicker, P. W., Beubler, E., Kukovetz, W.R. and Ritter, W. (1984) Dermatologica 169, (1), 51-56 14 Berg, D. and Plempel, M. (1984) Dermatologica 169, 3-10 15 Plempel, M. and Berg, D. (1984) Dermatologica 169, 11-18 16 Baloch, R. J., Mercer, E.A., Wiggins, T.E. and Baldwin, B.C. (1984) Proc. BCPC, Brighton 1984, Vol. 3, 893-898 17 Dixon, D. and Shadomy, S. (1978) J. Infect. Dis. 138, 245-248 18 Biichel, K. H., Draber, W., Regel, E. and Plempel, M. (1972) Drugs Made Ger. 15,

Receptor coupling to polyphosphoinositide turnover: a parallel with the adenylate cyclase system

79-94

19 Holt, R. (1972) Adv. Antimicrob. Antineoplast. Chemother. 1, 243-247 20 Yamaguchi, H., Hiratani, T. and Plempel, M. (1983) Drug Res. 33 (I) 4, 546-551 21 Plempel, M., Regel, E. and BLichel,K. H. (1983) Drug Res. 33 (I) 4, 517-524 22 Plempel, M. and Bartmann, K. (1972) Drugs Made Ger. 15, 103-120 23 Plempel, M. and Berg, D. (1984) GITSuppl. 4 (6), 27-30 24 Sch/ir, G. and Kayser, F.H. (1976) Chemotherapy 22, 211-220 25 Borelli, D. (1979) Postgrad. Med. J. 55, 657-661 26 Graybill,J. R. and Craven, P. C. (1982)in 22nd Interscience Conf. of Antimicrobial Agents and Chemother. (Philips, I. and Wise, R., eds), pp. 530, Academic Press 27 Heiberg, J. K. and Sveygaard, K.E. (1981) Brit. Med. J. 283, 825--829

specific G protein, GTP displaces GDP from the 0c subunit of the G protein which then p r o b a b l y dissociates into its c¢and ~y subunits, and the receptor affinity for agonists is decreased. Although the physiological relevance is d u b ious, [AIF4]- can also promote dissociation of G proteins apparently b y mimicking the y phosColin W. Taylor and Janet E. Merritt phate group of GTP. The subunits remain dissociated for as long as GTP is b o u n d to the 0~ subunit; Calcium-mobilizing receptors stimulate polyphosphoinositide hydrolysis, u p o n hydrolysis of this GTP by an catalysed by phospholipase C, with the formation of intracellular messengers intrinsic GTPase, the subunits that then control the cytosolic Ca t+ concentration and the activity of protein reassociate. Stable analogues of GTP (e.g. GTPyS and G p p N H p ) kinase C. Colin Taylor and Janet Merritt describe recent studies which suggest that a guanine nucleotide-dependent regulatory protein (G protein) couples are not hydrolysed b y this GTPase these receptors to phospholipase C, a finding that presents a striking parallel and they thereby prevent reassowith receptor regulation of adenylate cyclase. ciation of dissociated subunits. Two different G proteins (Gs and Gi), which differ in their c~ The initial response of m a n y cells coupling mechanism m a y share subunits (% and 0ci), mediate the to Ca2+-mobilizing receptor agonfeatures with receptor coupling to effects of receptors that stimulate adenylate cyclase. ists is the hydrolysis, catalysed or i n h i b i t adenylate cyclase acb y p h o s p h o l i p a s e C, of phosReceptor coupling to adenylate tivity. The JBysubunits of Gs and Gi phatidylinositol(4,5)bisphosphate are very similar, suggesting that Gs to inositol(1,4,5)trisphosphate [Inscyclase The adenylate cyclase signalling and Gi differ primarily in their 0c (1,4,5)P3] and diacylglycerol. Both subunits. The dissociated % subproducts have intracellular messystem consists of three essential unit stimulates adenylate cyclase senger functions 1, Ins(1,4,5)P3 components, a receptor, a guanine causes release of Ca 2+ from intranucleotide-dependent regulatory activity, b u t the mechanism cellular pools and diacylglycerol protein (G protein), and adenylate w h e r e b y dissociation of Gi leads cyclase2. Purification of the 6activates protein kinase C. Alto inhibition of adenylate cyclase though these products of phosadrenergic receptor, G proteins, activity is not entirely clear. Direct pholipase C activity have received and adenylate cyclase has been effects of 0ci are probably less much attention, the coupling of followed b y their reconstitution important than the effects of the receptors to p h o s p h o l i p a s e C has into a hormone-responsive sysfree jBy subunits which b y regulatonly recently attracted interest. In tem 3. These reconstitution experiing the availability of % may this review, we discuss recent ments, together with detailed relieve tonic stimulation of adenylevidence suggesting that this analyses of the complete system in ate cyclase activitys. native membranes 4 and of cells ~ Although the essential features genetically deficient in one or of receptor regulation of adenylate Colin Taylor is a Research Fellow at St Johns other component have p r o v i d e d cyclase are clear, some aspects College and the Department of Zoology, the following model of receptor remain subjects of debate. One Downing Street, Cambridge CB2 3EJ, UK. regulation of adenylate cyclase. such area of controversy is the Janet Merritt is a Postdoctoral Research Fellow Following b i n d i n g of its agonist, physical relationship between reat Smith, Kline and French Research Ltd., The Frythe, Welwyn, Herts, AL6 9AR, UK. the receptor associates with a ceptor, G protein, and adenylate ~ 1986,El~vierSciencePublisheP~B.V.,Amsterdam 0165-6147/86/$02.00