design of peptide hormone and neurotransmitter analogues

TIPS - June 1985

as a n e w class of cholestat ic agents w h i ch are active in bo th the rat and a n o n - h u m a n pr imate . The s ignif icance of these f i n d i n g s is at least twofold: (1) they clearly show that g lucu ron ide conjuga tes are no t a lways non tox ic e n d p roduc t s of d rug me tabo l i sm: a n d (2) they s t rongly sugges t that the de- creased hepa t ic excretory func t ion obse rved in p r e g n a n c y a n d in m e n and w o m e n treated w i th es t rogens may be du e to es t rogen D- r ing g lucu ron ide metabol i tes .

R e f e r e n c e s 1 Zimmerman, H. J. and Maddrey, W. C.

(1982) in Diseases of the Liver, 5th edn.

(Schiff, L. and Schiff, E.R., eds), pp. 621-692, Lippencott Co., Philadelphia and Toronto

2 Anderson, K. E. and Kappas, A. (1982) in Diseases of the Liver, 5th edn. (Schiff, L. and Schiff, E. R., eds), pp. 167-235, Lippencott Co., Philadelphia and Toronto

3 Kreek, M. J., Weser, E., Sleisenger, M. H. and Ieffries, G. H. (1967) N. Engl. J. Med. 277, 1391-1395

4 Meyers, M., Slikker, W., Pascoe, G. and Vore, M. (1980) J. Pharmacol. Exp. Ther. 214, 87-93

5 Vore, M., Montgomery, C. and Meyers, M. (1983) Drug Metab. Rev. 14, 1005- 1019

6 Slikker, W., Vore, M., Bailey, J.R., Meyers, M. and Montgomery, C. (1983) J. Pharmacol. Exp. Ther. 225, 138-143

7 Vore, M., Hadd, H. and Slikker, W. (1983) Life Sci. 32, 2989-2993

8 Smith, E. and Kellie, A. (1967) Biochem. I. 104, 83-94

259

9 Lucier, G. W., and McDaniel, O.S. (1977) J. Steroid Biochem. 8, 867-872

10 Wright, K., Collins, D. and Preedy, J. (1979) Steroids 34, 445-457

11 Williams, M. C. and Goldziehr, J. W. (1980) Steroids 36, 255-282

12 Raitano, L. A., Slikker, W., Hill, D. E., Hadd, H.E., Cairns, T. and Helton, E.D. (1981) Drug Metab. Disposition 9, 129-134

13 Erlinger, S. (1982) in Diseases of the Liver, 5th edn. (Schiff, L. and Schiff, E.R., eds), pp. 93-118, Lippencott Co. Phila- delphia and Toronto

14 Meier, P. J., St. Meier-Abt. A., Barrett, C. and Boyer, J. L. (1984) J. Biol. Chem. 259, 10614-10622

15 Brock, W. J. and Vore, M. (1984) J. Pharmacol. Exp. Ther. 229, 175-181

16 Brock, W. J., Durham, S. and Vore, M. (1984) J. Steroid Biochem. 20, 1181-1185

17 Adinolfi, L. E., Utili, R., Gaeta, G. B., Abernathy, C.O. and Zimmerman, H. J. (1984) Hepatology 4, 30--37

Design of peptide hormone and neurotransmitter analogues Victor J. Hruby

Peptides are being found to be increasingly important as hormones and neurotransmitters, and much research is directed towards elucidating the physiological roles of these natural products in pain, reproduction, maintenance of blood glucose levels etc. In this review, Victor H r u b y describes some of the conformational approaches that have led to significant insights into the chemical and physical properties of these compounds and so to the rational design of analogues with new and specific properties.

Efforts to u n d e r s t a n d the phys i - ca l -chemica l basis for pep t ide h o r m o n e a n d n e u r o t r a n s m i t t e r biological activit ies, a n d to develop this class of c o m p o u n d s as useful pharmacologica l agents has b e e n h i n d e r e d by the genera l p roper t ies of m a n y of these com- p o u n d s . These include: (1) rap id b iodeg rada t i on ; (2) mul t ip le acti- v i t ies at d i f ferent receptors; (3) shor t d u r a t i o n of biological response ; (4) h igh degree of conforma t iona l f lexibil i ty; and (5) low oral activity.

These proper t ies are those ex- pected of molecules wh i ch na tu re has evolved to serve as biological swi tches a n d / o r media tors of in tercel lu lar c o m m u n i c a t i o n . H o w - ever, recent s tudies , have s h o w n that these proper t ies n e e d no t be in t r ins ic for pep t ide hor-

Victor J. Hruby is Professor of Chemistry and of Biochemistry, Department of Chemistry, University of Arizona, Tucsan, AZ 85721, USA.

m o n e a n d neu ro t r ansmi t t e r analogues. Indeed , by careful ut i l iza- t ion of conforrnat ional cons t ra in ts a n d cons ide ra t ion of topological a n d l ipophi l ic propert ies , pep t ide ana logues can be ob t a ined wh ich comple te ly overcome one or more of these ' u n d e s i r a b l e ' propert ies . This rev iew descr ibes some of the conformat iona l approaches wh ich have led to pep t ides w i th h igh receptor specificity, p ro longed in- vivo activity, s tabi l i ty to proteoly- tic e n z y m e s and s igni f icant oral activity.

The basic hypothes i s of this approach is that the conforma- t ional proper t ies of pept ides , and their abi l i ty to a s sume different conformat ions , are in t imate ly related to their biological activities. Most of these activit ies involve in terac t ions wi th m e m b r a n e - b o u n d receptors wh ich are part of complex macromolecular systems. Biological i n fo rma t ion t ransfer for these c o m p o u n d s general ly in - volves three closely related events :

(1) receptor r ecogn i t ion or b i n d - ing; (2) biological t r ansduc t ion (act ivat ion of the biological response) lead ing to agonis t or an tagonis t ( inhib i tory) activity; and (3) d issocia t ion of the hor- m o n e from receptor 1~. We have hypo thes ized 4-7 that each of these three steps ut i l ize different conformat ional and s tructural p rop- erties of the h o r m o n e d u r i n g its in terac t ion wi th the receptor. Thus proper t ies of the h o r m o n e related to potency, to efficacy (agonist , an tagonis t , mixed agon- i s t -an tagonis t , etc., activity), and to du ra t ion of response can, in pr inciple , be separately modi f ied by appropr ia te m a n i p u l a t i o n of the pept ide conformat ion and structure.

In this context, a ra t ional approach to the des ign of pep t ide ho rmones wi th specific biological proper t ies will u t i l ize specific conformat ional and topographical de- s ign in relat ion, if possible , to several different receptor systems. If those different features of structure and confo rmat ion that are critical for receptor recogni t ion , t ransduc t ion , or reversal of response at a specific receptor are carefully cons idered , then conformat ional ly-res t r ic ted ana logues wi th disparate biological activit ies can be prof i tably examined for their conformat ional and topological proper t ies u s i n g physical me thods such as NMR spectroscopy. Results from these, and s t ruc ture-b io logica l activity, s tudies can then be used to develop a specific mode l for the relat ion- sh ips of s t ructure and conforma- t ion to in teract ion wi th a par t icular

~) 1985, Elsevier Science Publishers B.V., Amsterdam 01~5 6147/85/$02.00

260 TIPS - June 1985

(a)

(b)

CH2-2 i I

NH2CHCO-Tyr-ne-Gln-Asn-Cys-Pro-Leu-Gly-NHs ox~rtoetn

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp

HO-Cys-Ser-Thr-Phe-Thr-Lys

somatos tat tn

(c) Ac-Ser-Tyr-Ser-Met-Glu-His

Phe H2N-Val-Pro-Lys-Gly-Trp-Arg /

~-melanotropin

(d) H-Tyr-D-NHCHCO-Gly-Phe I t

(CHz)4 NH t I

NI'-I~ HO2C-CHCH2CH(CHsh

lz,~"~.,a, eptua.tn

(e) H-Tyr-NHCH2CO-Gly

CHs I \ Phe S /

CH2 \

HO2C-CH-NH

I M ~ u d U l Fig L Examples of conformationa/ restrictions /n peptic/as.

~CHs)s-S I NH2CHCO-Tyr-ne-Gln-Asn-Cys-Pro-Leu-Gly-NHz

r--- Cys-Phe-D-Trp

t__ Cys-Thr - Lys

(N-Me)Ala-Phe-D-Trp

- - I I Phe-Thr - Lys

Ac-Ser-Tyr-Ser-Cys-Glu-His

H~-Val-l~o-L~-G~-'r~-/~xg - Phe

I-t-Tyr-I~NHCHCO-GIy-Phe I I

(G~t2)4 NH j I NH-CO ~ CH~I2Ct'I(CI-~

H-Tyr-D=NH-CH-CO-GIy I

,C(CI"hh S

\ S /

c(cI-hh /

HO2C-CH-NH-D-Phe

receptor. Such models are tested by synthesis of analogues with specific conformational, topological and structural propert ies.

Various aspects of the development of this approach have been discussed 4-18 and a few recent examples i l lustrating some of the general pr inciples which are emerging from these studies are descr ibed below.

C o n f o r m a t i o n a l r e s t r i c t i o n s o f cyclic pep t ides

Oxytocin ( H - C y s - T y r - I l e - G l n - Asn-Cys-Pro--Leu--Gly-NH2) a pep t ide hormone best known for its effects on uter ine contraction and milk ejection, possesses a disulf ide-containing 20-membered ring. Further conformational restriction of this med ium size r ing has been accomplished by replacing a Cys 1 res idue (side chain = -CH2-S-) wi th a pencill- amine (side chain = - C ( C H 3 ) 2 - S - - ) res idue (Fig. 1,a). The conformational consequences of such a restriction are two-fold: (1) due to steric effects, it renders the disul- fide d ihedra l angle to greater than

100°; and (2) the t ransannular effect of the geminal d imethyl group in the twen ty -membered r ing restricts both the backbone and s ide-chain conformations 4. The biological consequences of these structural restrictions is that an oxytocin inh ib i tor (antagonist) is obtained. We have in terpreted these results and other studies, especially by Manning and co- workers, to suggest that the receptor antagonis t activities of this and related penici l lamine-con- ta ining oxytocin analogues are specifically related to the conformational restrictions that are pre- sent4,~8.

Subsequent s tudies in several laboratories 4,6,s,9,12,16 have suggested that appropr ia te conformational restrict ions wi th in med ium size pep t ide rings, including r ing size reduction, can lead to insights into the differential structural and conformational requirements for receptor recognit ion (binding) and transduction for several peptides. For example, Veber and co- workers s,17 directed their efforts at obta in ing small, conformation-

al ly-constrained analogues of somatostat in which re ta ined the potency of the nat ive pept ide , but which would be biologically stable against degradat ion. A series of increasingly smaller r ing systems, and bicyclic r ing systems which still d i sp layed high biological potency were examined (Fig. lb) and eventually a cyclic hexapept ide 18-membered r ing system (Fig. lb) that had the same biological potency as the nat ive pept ide , but which is less than 50% the molecular weight of somatostat in was identif ied. Some of these biologically potent pep- t ides were found to be biologically stable and orally active. In the course of these invest igat ions biophysical and conformational insights from NMR spectroscopy and from computer -based molecular model ing were used to direct the des ign of new analogues.

It should be noted that conformational restriction by reduct ion in r ing size is not universal ly applicable. For example, early work on oxytocin (see Ref. 18)

TIPS - June 1985

showed that reduct ion in r ing size from a 20-membered to a 19- membered r ing led to drastically reduced biological activity. Inter- estingly, an increase in r ing size from a 20- to a 21-membered r ing also led to drastically reduced potency. Similar results have been noted in our laboratory for cyclic melanotropin analogues (vide infra). It thus appears that there is an op t imum ring size for inter- action of a pep t ide hormone or neurot ransmit ter wi th its receptor, and this may vary be tween one receptor and another for a part icular hormone, as well as be tween different hormones.

Conformational restrictions: l inear peptides to cyclic peptides via side chain - s ide chain covalent attachment

Favorable conformational res- tr ictions of l inear pep t ides are often difficult because of the problem of de te rmin ing a priori which of the many poss ible conformations is impor tan t to biological activity. However , several approaches have proven to be quite useful.

N~-Methyl (or other alkyl groups) and C~-methyl subst i tu- tions restrict the conformational space available to the modi f ied res idue and can have more exten- sive topological effects in some c a s e s 9,12,16. S u c h structural modif i - cations are general ly most useful for provid ing insight into the impor tance of conformation for specific res idues to b ind ing and transduction. Introduct ion of a D- amino acid res idue in the place of the cor responding L-amino acid res idue can often provide insights about those por t ions of the pep- t ide where a reverse turn conformat ion is likely to be of significance for the biological activity of the pep t ide ~2. For example, the super potency and pro longed in- vitro (hours to days) and in-vivo (days to weeks) activity of [Nle 4, D-Phe7]-~c-MSH (melanocyte-stim- ulat ing hormone, Ac -Se r -Tyr - Ser-Nle-Glu-His--D--Phe--Arg-Trp- Gly-Lys-Pro-Val -NH2) in the frog skin and melanoma adenylate cy- clase assays systems led us to postulate 6,9 that a reverse turn conformation might be an important feature of the 'biologically- active' conformation of ~-melanotropin. The conformational model deve loped from these s tudies led to the des ign and synthesis of a

cyclic analogue to test the model (Fig. lc). The compound prepared, [Cys 4, CyslO]-oc-MSH, turned out to have superpoten t activity in the frog skin assay system and high enzymatic stability.

Pseudoisosteric covalent cyclizat ion

This latter analogue illustrates the appl icat ion of another conformational and topographical approach that may have general significance. We have termed this k ind of structural modif icat ion 'pseudoisos ter ic covalent cyclization'. The basic idea in this approach is to examine the conformational and topological properties likely to be impor tant for hormone- recep to r interact ion and from this model de termine whe the r sequence - sepa ra t ed s ide g roups are in close proximi ty . If this is the case, a specific topologica l feature can be s tabi l ized by covalent chemical a t tachment of the two side chain moieties. [Cys 4, Cysl°]-~c-MSH illustrates this principle as follows. Conformational models suggested that the Gly 1° and Met 4 residues were in close spacial proximity (Fig. lc). The Cys 4, Cys 1° modifi- cation (side chains = -CH2-S--S-- CH2-) is pseudoisoster ic to a Met 4 (side chain = -CH2-CH2-S--CH3) and Gly 1° (side chain = -H). The modif icat ion, of course, converts a l inear pep t ide into a pseudoisosteric cyclic analogue (Fig. lc). This pr inciple can be appl ied to almost any side chain group found in pept ides .

Side chain-backbone cyclization Another approach which prom-

ises to f ind general appl icat ion is s ide-chain to backbone covalent cyclization 1°-12,2°. An excellent i l lustration of this approach can be found in the enkephal in analogues of Schiller and co- workers 1°,2° in which [Leu]enkephal in (H-Tyr-Gly- -Gly-Phe-Leu- OH) was converted to a series of cyclic analogues of which H - T r y - c[ -NLD--Lys-Gly-Phe-Leu-] is an example (Fig. ld) . Covalent attachment of the e-amino group of D-Lys 2 res idue to the C-terminal backbone carboxylate group of Leu s p roduced a cyclic 16-membered r ing analogue of enkephal in that has high potency and sig- nif icant mu (~) receptor selectivity. Other examples of this approach to other biologically active

261

pep t ides have been reported by Chipens and co-workers 11 and others 12.

Combinations and permutations Two or more of the above ap-

proaches can, of course, be com- b ined to obta in new insights. For example, the pseudoisoster ic covalent cyclization approach was used in conjunction with the geminal d imethyl t ransannular sub- s t i tut ion effect in [Met]enkepha- lin to form [D-Pen 2, D-PenS]en - kephal in a cyclic 14-membered r ing structure wi th two geminal d imethyl groups wi thin the r ing (Fig. le). This compound has ex- t raordinary delta (6) receptor selectivity and high biological stab- il i ty 21. These results illustrate another rather common effect of covalent conformational restriction in pep t ide hormones and neurotransmitters , namely the development of high receptor selecti- vi ty for the analogue in compari- son with the more flexible native hormone.

[ ] [ ] [ ]

The above results, and others which could be cited 12,16 suggest that careful considerat ion of class- ical s tructure-act ivi ty data in terms of conformational and topological relat ionships to biological activity can provide a powerful, rational approach to the design of pept ide hormones and neurotransmitters wi th specific biological properties. Covalent stabiliza- tion of appropr ia te conformational and topographical structural features can provide significant new insights into the chemical- physical basis of information transfer for these important compounds. It can be predicted that the development of appropria te synthetic and conformational approaches to obtain such compounds will have an important impact on the development of pept ide drugs.

References 1 Van Rossum, J.M. and Ariens, E.J.

(1962) Arch. Int. Pharmacodyn. Ther. 136, 385-413

2 Rudinger, J. (1971) in Drug Design, Vol. 2 (Ariens, E J., ed.), pp. 319--419, Academic Press, New York

3 Schwyzer, R. (1973) in Peptides 1972 (Hanson, H. and Jakubke, H. D., eds), pp. 423-436, North-Holland, Amster- dam

262

4 Hruby, V. J. (1981) in Topics in Molecular Pharmacology (Burgen, A.S.V. and Roberts, G.C.K., eds), pp. 99-126, Elsevier/North-Holland, Amsterdam

5 Hruby, V.J., Knittel, J.L., Mosberg, H. I., Rockway, T. W., Wilkes, B C. and Hadley, M.E. (1983) in Peptides 1982 (Bl;iha, K. and Malon, P., eds), pp. 19- 30, de Gruyter, Berlin

6 Hruby, V. J. (1984) in Conformationally Directed Drug Design. Peptides and Nu- cleic Acids as Templates or Targets (Vida, J. A. and Gordon, M., eds), pp. 9-27, ACS Symposium Series, 251, Amer. Chem. Soc., Washington, D.C.

7 Hruby, V.J. and Hadley, M.E. in XVIIIth International Solvay Conference on Chemistry (van Binst, G., ed.) (in press)

8 Veber, D. F. (1981) in Peptides: Synthesis - Structure - Function (Rich, D. H. and Gross, E., eds), pp. 685--694, Pierce

Chemical Co., Rockford, IL 9 Marshall, G. R., Gorin, F. A. and Moore,

M. L. (1978) Annu. Rep. Med. Chem. 13, 227-238

10 Schiller, P. and DiMaio, J. (1983) in, Peptides: Structure and Function (Hruby, V. J. and Rich, D. H., eds), pp. 269-278, Pierce Chemical Co., Rockford, IL

11 Chipens, G.I., Krikis, A.J. and Polevaya, L. K. (1979) in Biophysical and Biochemical Information Transfer in Rec- ognition (Vassileva-Popova, J.V. and Jensen, V., eds), pp. 23-48, Plenum Press, New York

12 Hruby, V. J. (1982) Life Sci. 31, 189-199 13 Hruby, V. J., Krstenansky, J. and Cody,

W. L. (1984) Annu. Rep. Med. Chem. 19, 303-312

14 Schwyzer, R. (1977) Ann. N.Y. Acad. Sci. 297, 3-24

15 Spatola, A. F. (1981) Annu. Rep. Med. Chem. 16, 199-211

TIPS - June 1985

16 Vida, J. A. and Gordon, M., eds (1984) Conformationally Directed Drug Design. Peptides and Nucleic Acids as Templates or Targets, ACS Symposium Series No. 251, Amer. Chem. Soc., Washington, D.C.

17 Veber, D. and Saperstein, R. (1979) Annu. Rep. Med. Chem. 14, 209-218

18 Hruby, V. J. in Biochemical Actions of Hormones (Litwack, G., ed.), Academic Press, New York (in press)

19 Sawyer, T.K., Hruby, V.J., Hadley, M. E. and Engel, M. H. (1983) Am. Zool. 23, 529-540

21 Schiller, P. W. (1984) in The Peptides, Vol. 6 (Udenfriend, S. and Meienhofer, J., eds), pp. 219-268, Academic Press, New York

21 Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I., Galligan, J. J. and Burks, T. F. (1983) Proc. Natl Acad. Sci. USA 80, 5871-5874

Brain Slices and their pitfalls Brain Slices

edited by R a y m o n d Dingledine, P l enum Press, 1984. US$50.00 (xvi i i + 442 pages), I S B N 0 306 41437 6

The term 'brain slice' is defined by the editor, Raymond Dingle- dine, as thin (100--700 ~m) sec- tions prepared from adult mam- malian brain regions. The title of this book might perhaps be thought deceptive by those phar- macologists whose tools of trade are other than microelectrodes and yet who have used brain slices, as it is a imed almost entirely at electrophysiologists. There is however an excellent introductory chapter by Llinas on the various ionic currents to be found in different regions of the neuron, which is sufficiently well explained to be understood by those who have opened the book out of general interest. He empha- sises the contr ibut ion of various t issue slices in vi tro to the development of knowledge of these currents, al though of course, the basis of the subject was laid down well before the age of the slice.

The book continues wi th ten chapters on the use of slices from different brain areas for electrophysiological studies. Five of these are concerned with the hippocarnpus where a large pro- port ion of slice work has been done, partly for reasons of slice

survival, and partly because of intrinsic interest in hippocampal function. The range of chapters on the h ippocampus has been well chosen to illustrate the diversity of possible studies on brain slices. Turner and Schwartzkroin discuss the application of models of pass- ive and active neuronal properties to the hippocampal slice. The value for different applications of network, cont inuous cable, com- partmental and neuronal circuit

"With special ingredients from some of the top gourmets."

such plastic events as long term potentiat ion. The impressive quant i ty of prel iminary studies which they have performed to examine the feasibility of voltage- c lamping synaptic events at different distances from the soma are

a caveat to anyone thinking of embarking on similar analyses of other synaptic populations. models is considered in turn, and in the next chapter modell ing is also uti l ised by Johnston and Brown. These authors elegantly examine the problems and possi- bili t ies afforded by the hippocampal slice for quantal analysis of synaptic events or 'microphy- siology'. One of their goals is to examine the synaptic basis for

A different approach to examin- ing the basis of hippocampal synaptic plasticity is repesented by the chapter of Lynch, Kessler and Baudry who have made several heroic attempts to correlate long term potentiat ion with biochemical changes in hippocampal slices. The problems associated with this type of study are as great as those encountered by Johnston and Brown, although at the opposite end of the spectrum. How to stimulate enough of the slice to give any biochemical changes a chance to show up, and how to use a small enough slice that the biochemical changes will be in a reasonably homogeneous popula- tion of synapses are two of the problems they have encountered. Again, this chapter is an object lesson to anyone about to embark upon such combined biochemical and electrophysiological studies, that although the concepts may be simple, the practicalities will abound with pitfalls.

The chapter by Dingledine is a comprehensive account of the different pathways, both intrinsic and afferent to the h ippocampus util ising all the major classes of neurotransmitter. The effects of

design of peptide hormone and neurotransmitter analogues

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