TiPS - September 1991 [Vol. 121

New influences of biotechnology in drug discovery: is the rat an endangered species? Although biotechnologicai prod- ucts per se have not been the therapeutic and commercial suc- cess that was predicted in the optimistic era of the 1970s and 19809, biotechnology is influ- encing drug development in a fun- damental way. An international symposium’ dedicated to this theme discussed the powerful new screening methods and new types of target offered by biotech- nological techniques.

The impact of biotechnology on the pharmaceutical industry is changing (Adrian Hobden, Glaxo Group Research, Greenford). ‘Traditional’ biotechnology in the form of antibiotic production is still much more valuable in commercial terms than ‘modem’ biotechnology involving genetic engineering: the antibiotic market is worth over $10 billion annually, or about 5% of the total pharma- ceutical market. By contrast, the total sales from the much-heralded revolution through therapeutic proteins still lags well behind any one of the seven current ‘small molecule’ blockbusters (e.g. raniti- dine, captopril, nifedipine), each of which had sales valued at over $1 billion in 1990. However, there are several areas where molecular biology and genetic engineering are influencing drug discovery in a more fundamental way. In par- ticular, these techniques have provided unique assay tools, humanized antibodies, new in- sight into disease mechanisms and novel functional assays, in- cluding cloned and expressed human receptors and transgenic apimals.

Cloning of human receptors for neurotransmitters and expressing them in suitable host cells makes it possible to taset drugs to single human Droteins (Paul Hartin. Neuroge&tic Corpbration, Par& mus, New Jerse,v). Hartig pre-

sented evidence that human 54iT1o and 5-HTz receptors can be SUCC~SS~IIU~ expressed in non- human, non-neuronal cell lines (mouse fibroblast cells). These cells offer the advantage of a ‘zero background’ (i.e. they express no monoamine receptors them- selves), as well as being easier to hanale. The fact that human re- crpt0rs expressed in a mouse cell have the pharmacological profile of the human rather than the mouse receptor emphasizes that it is the primary structure itself, and not the environment, that dictates properties. The drawback of this system is that functional re- sponses are not easy to determine, and a second level of screening to determine intrinsic efficacy is required.

Non-mammalian systems offer greater potential efficiency for high-throughput screening. Hob- den described the system that has been developed by the Lefkowib and Caron group at Duke University (see Ref. 1 and Fig. 1). This group exploited the signal transduction system used by the G protein-coupled phero- mone receptors in the yeast Sac- charonyces cereuisiae to develop a host in which responses to agonist binding to a transfected receptor can be measured calorimetrically. They co-transfected a modified human &-adrenoceptor gene, under the control of the galactose- inducible GAL1 oromoter (to obtain high expre’ssion It&s), with the mammalian G-protein subunit Gr~, into a strain-of the yeast in which a pheromone- responsive FUSl gene promoter had been fused with a reporter gene (fi-galactosidase) and stably integrated into the genome. Al- though it has not yet been ‘indus- trialized’, drug companies are interested in this system because agonist binding activates fi-galac- tosidase, and this activity can be translated into a simple colocr- change assay that is adaptable to

317

automation and large numbers of tests.

Similar cloning approaches can be used to provide enzymes or other potential theraperitic ta;geb for other high-throughput assay systems (Louis N&bet, Xenova, Slough).

However, one caveat to the use of single human proteips as the principal screening system came from the work of Sir James Black (James Black Foundation, Lon- don), in which the subtle differ- ences in effect of a compound on tissues from different species could illuminate key structure- activity relationships. Black argues that experiments on iso- lated fragments of a normally complex and interacting system could fail to reveal more subtle ways to control novel therapeutic targets.

Cloning and DNA sequencing reveals that the closest relatives of a particular receptor may often be a receptor in the same structural superfamily but for a different neurotransmitter. Subtypes of re- ceptors for the same transmitter frequently belong to structurally distinct dasses. The implications of this for drug design have yet to be exploited. This technology can also answer questions relating to species differences. Hartig reported the cloning of the rat 5-HTls receptor which he has shown to be -90% homologous with the human 5-HT,o receptor. Differences in crucial amino acids are responsible for the differences that have been observed in pharmacology.

Cloning techniques have not only proved useful in revealing differences between receptor sub- types, but they are also opening up various parts of the intra- cellular signalling systems as possible targets for selective drug action. This is because the major enzymes involved in the for- mation and de&r&on of &cond messengers have been shown to exist as different subtypes, which also may have several isomeric forms (Paul England, SmithKline Beecham, Welwyn). This has re- opened the possibility of drugs acting selectively on tissues con- taining particular subtypes of, for example, phosphodiesterases or protein kinases, although selec- tive cell penetrability may also be possible to achieve. Large

318 TiPS - September 2991 /Vol. 121

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numbers of inhibitors of phos- phodiesterases and, more recently, protein kinase C (Ref. 2) are being characterized. In addition to the well-known second messengers (cyclic nucleotides, inositol phos- phates, and diacylglycerol), there may be equivalent systems associ- ated with tyrosine kinase-linked receptors for growth factors. Selec- tive agents against tyrosine kinases are now known, and in- clude the tyrphostins3. As many receptors couple to their second messenger signalling system via G pr+eins, which can exist in mul- tiple isomeric formsJ, selective

therapeutic agents may be able to act on G proteins (Nigel Pyne, University of Strathclyde). This is attractive in disease states such as hypothyroidism and asthma in which C proteins in affected tis- sues are abnormal. Current strat- egies involve compounds that exert negative intrinsic activity towards the G protein (i.e. they block the ‘empty receptor’ effects), which perturb receptor-G protein interaction, or which alter GTP- GDP exchange in the guanine nucleotide-binding domain. Re- cent results indicate that some phasphodiesterase inhibitors can

also affect the guanine nucleotide- binding domain of G proteins.

Cloning techniques have also been used to investigate the func- tional sites on biologically active proteins. Site-directed mutagen- esis has been used to define the important residues governing the interaction of snake curaremimetic toxins with nicotinic acetylcholine receptors (And& Mnez, CEN Saclay, France). These techniques can be used to introduce new functions into the exceptiom4ly stable structural framework pm- vided by the polypeptide toxins from animal venoms. Recent structural determinations of scorpion toxins5 reveal how dif- ferent biological functions can be provided using a common S~UC- tural motif. Further exploitation of structural information from small protein toxins was suggested as a means to design highly selective ligands for specific membrane ion channels (Alan Harvey, Strath- Clyde University).

Advances in antibody tech- nology were described by Greg Winter (Cambridge Centre for kotein Engineering). Libraries of antibody genes from immunized mouse B cells can be prepared using the polymerase chain re- action, antibodies expressed on the surface of bacteriophage by fusion to a minor coat protein, and the appropriate phage enriched by affinity chromatography. The potential exists for creating anti- bodies with new prop&es with- out human or animal immuniz- ation A different approach to the engineering of cells to provide sophisticated properties in exper- imentally convenient systems is to immortalize the target mam- malian cell using immortalizing viral genes (Caroline MacDonald, University of Strathclyde). Kidney, liver and macrophage cells have been successfully im- mortalized using these tech- niques.

Although gene cloning has pro- vided a vast amount of infor- mation about the amino acid sequences of potential drug tar- gets, the details of three-dimen- sional structure has been unable to keep pace. X-ray crystal- lography is always likely to be slower than a pdymerase chain reaction. However, the increase of power of readily accessible cam- puters could provide a solution, if

TiPS - September 1991 /Vol. 121 319

the problem of predicting protein structure from primary sequences could be solved. Different ap- proaches were described by Graham Richards (Oxford Univer- sity), Barry Robson (Proteus Mol- ecular Design) and Mark Dufton (Strathclyde University). Al- though progress is being made, not enough examples have been

completed to give a positive verdict.

ALAN HARVEY

References 1 King, K., Dohlman, H. G., Thomer, J.,

Caron. M. C. and Lefkowitz, R. J. flY!:

Srhce 250. 121-123 2 Tamaoki. T. and Nakano, H. (1990) Eio/

Tccl~nolwu 8. 732-735 . . . 3 Levitzki, A. and Gilon, C. (1991) Trends

Plmrmrool. sci. 12, 171-174 4 Boumc H. R.. Sanders, D. A. and

McCormick, F. (1991) Nntwr 349, 117-127

5 Bontems, F. et RI. (1991) Err. /. Biockrm. 1%. 19-20

6 Winter, C. end Milstein, C. (1991) NP~IVC 349,29>299

Noradrenalin~ATP co-transmission in the sympathetic nervous system

Responses to sympathetic nerve stimulation that are resistant to adrenoceptor-blocking drugs have long puzzled pharmacologists. The most likely explanation for this phenomenon now is co-trans- mission by several transmitters. Peptides such as NPY and galanin, and also amines such as dopamine and adrenaline, may accompany noradrenaline as transmitter sub- stances in postganglionic sym- pathetic neurons.

The idea that ATP is a co- transmitter of noradrenaline has developed since the early 1970s. The evidence is so far largely restricted to excitatory responses in smooth muscle (Table I), re- sponses that exogenous ATP is known to elicit via the Pzx sub- type’ of purinoceptor. This article summarizes the evidence for the hypothesis of nomdrenaline_ATP co-transmission and discusses some unresolved issues.

Evidence from postjunctional deseneitization or blockade

a&Methylene-ATP (a$- MeATP) is a metabolically stable ATP analogue that first activates and then desensitizes Pzx purino- CeptorS’. Arylazidoaminopro- pionyl ATP (ANAPP3), which co- valently binds to P2x receptors after irradiation in the presence of tissue, similarly causes receptor activation and then blockade’. Suramin, a drug used in the treat- ment of trypanosomiasis since 1920, has recently been shown to block P2x receptors z4. All three compounds attenuate adrenocep-

tor antagonist-resistant cnon- adrenergic’) responses to sym- pathetic nerve stimulation in the frog heart and in several mam- malian smooth muscle organs, particularly the vas deferens and various blood vessels (Table I).

Blockade of the non-adrenergic responses can be obtained with- out any change in either the response to exogenous noradrena- line or the release of noradrena- line. Both excitatory junction potentials and contractions (or their non-adrenergic components) are attenuated in smooth muscle.

The common, selective antagon- ist effects of a,@MeATP, ANAPP3 and suramin demonstrate that the non-adrenergic responses are me- diated by Put receptors and, hence, purinegic. Since purinergic as well as adrenergic components are blocked by Chydroxydopa- mine and guanethidine, com- pounds that act selectively on catecholamine-containing axons, both the noradrenaline and the purinergic transmitter must come from the same neurons, indicating co-transmission rather than paral- lel transmission from purely nor- adrenergic and pilrely purinergic fibres. The majo! purinergic trans- mitter appears to be ATP, but other contenders exist (see below).

Figure 1 shows the pharmaco- logical dissection of adrenergic and purinergic components of muscle contraction at a high degree of temporal resolution. In the mouse vas deferens, the twitch elicited by the first pulse in a train of low-freqltency pulses is

often biphasic and higher than subsequent twitches, where a marked decrease of the second, slow phase obscures the biphasic character (control trace ‘C’ in Fig. la and lb). The aI-adrenoceptor antagonist prazosin abolished the (large) slow phase of twitch 1 as well as the (small) slow phase of twitches 2 and 3, while leaving the rapid phase almost intact (‘P’ in Fig. la). Higher concentrations of prazosin failed to cause any ad- ditional change. Thus, the slow phase is adrenergic, but the rapid phase is resistant to praxosin and therefore non-adrenergic. Its purinergic nature is supported by the effect of suamin, which com- pletely blocked the prazosin-re- sistant contraction (‘P + S in Fig. la). The reverse addition yielded the opposite sequence: when given first, suramin selectively abolished the rapid phase (‘S’ in Fig. lb), and prazosin then elim- inated the remaining slow phase (‘S + P’ in Fig. lb). Desensi- tization by a$-MeATP produced a twitch pattern identical to that obtained in the presence of suramin’. The fundamentally dif- ferent fate of the purinergic and adrenergic components in the course of a pulse train - the maintenance of the purinergic twitches versus the precipitous fall of the adrenergic twitches - is discussed below.

Evidence from ATP overflow ln postganglionic sympathetic

axons, ATP is co-stored with nor- adrenaline in small and large synaptic vesicles”. After preincu- bation with 13H]adenine or 13H]adenosine, sympathetic nerve stimulation releases tritiated compounds in, for example, blood vessels and the vas deferens. The overflow of endogenous ATP has been determined recently using the luciferin-IuciferaSe method, or high pressure liquid