ture determination and synthesis of the polypeptide and protein hormones, but much remains to be done (286, 287). Therefore, it follows that work on the design, synthesis, and evaluation of analogues of these hormones, an area of great potential interest, has just begun. Modification of the polypeptide and protein hormone structures by synthesis or by chemical or enzymic methods has been employed primarily to determine structure-activity relationships and to elucidate which amino acid or peptide residues are essential for a given hormonal effect. Although most chemically altered hormones show only reduced activity, certain of these derivatives possess inhibitory properties. Structure-activity relationships of the oxytocin and vasopressin molecules serve as the best example of the status of research in this field.
These inhibitory hormone analogues are considered in two general categories: inhibitors of hormone activity and inhibitors of hormone-inactivating systems (in effect, potentiators of hormone activity).
A. Inhibitors of Hormone Activity
The inhibitors of hormone function may be further classified as syn
thetic analogues of known structure and uncharacterized derivatives prepared by chemical modification of the parent hormones.
1. CHEMICALLY CHARACTERIZED INHIBITORS
a. Oxytocin and Vasopressin Analogues. The cyclic octapeptide hor
mones, oxytocin and vasopressin, isolated from the posterior pituitary and classified as neurohypophyseal hormones, have been synthesized.
These hormones have similar chemical structures and (to greatly differing degrees) similar biological properties. The synthesis and biological evalua
tion of analogues have helped to elucidate the biological function of certain amino acid components of these molecules.
The complete structure of oxytocin ( I X ) is written in abbreviated form ( X ) according to the Brand and Edsall system (4). The structures of the vasopressins are the same as ( X ) except that phenylalanine replaces isoleucine, and arginine or lysine replaces leucine.
CHa
N H2
j
C H3C H a C H C O N H C H C O N H ^ H C H C H a C H s Cy — T y r — l i e u
S
m
S N HaC ^ C H N H C O C H N H C O C H C H a C H a C O N ^ (Jjy À s p - G l u- N H ,
(j!0 C H2C O N H a P r o — L e u — G l y —N H a C H2— N v
j C H C O N H C H C O N H C H a C O N H a
C H2C H2/ CH2CH(CHs)2
( I X ) ( X )
Certain analogues and homologues of the oxytocin molecule inhibit the action of oxytocin or of vasopressin. A number of analogues of oxytocin (288-295) and of vasopressin (296) and "hybrids" of the two hormones (297j 298) have been synthesized. A convenient classification and sum
mary of the structures and biological activities of these compounds has been proposed by Acher (299). The analogues are classified as derivatives of oxytocin by indicating in the prefix the nature and position of the substituted residues. In general, structural changes in the oxytocin
mole-cule result in reduced oxytocic activity and in many instances increased pressor activity.
Two chemically defined analogues of oxytocin inhibit hormonal ac
tivity. Isoglutamine-4-oxytocin ( X I ) inhibits the pressor activity of administered vasopressin in the anesthetized rat (289). This compound, which may be regarded as a structural analogue of oxytocin or of vaso
pressin, possesses no hormonal activity itself and may act as an inhibitor by replacing vasopressin at the binding site. In preliminary experiments, ( X I ) also appeared to show some inhibition of the action of oxytocin on the rat uterus (289).
CyS · T y r · lieu. Iglu ( N H2) · Asp ( N H2) · CyS · Pro · Leu · Gly ( N H2)
1 2 3 4 5 6 7 8 9
( X I )
The oxytocin homologue ( X I I ) with one supplementary tyrosyl residue inhibits the action of oxytocin on the isolated uterus of the rat and the rabbit and in the avian depressor assay (300, 300a). It is of interest that the
CyS · T y r · T y r · lieu · Glu ( Ν Η2) · Asp ( N H2) · CyS · Pro · Leu · Gly ( N H2)
( X I I )
cyclic disulfide rings in the octapeptide ( X I ) and the nonapeptide ( X I I ) are both larger than those in oxytocin and vasopressin (289). l-(Hemi-homo-cystine)-oxytocin, an oxytocin analogue in which the half-cystine residue of ( I X ) bearing the free amino group is replaced by a half-homocystine residue, has been synthesized and is reported to inhibit the action of oxytocin in the avian depressor assay to a very slight degree (300b).
6. Angiotensin Analogues. Angiotensin (formerly known as hypertensin or angiotonin) is a polypeptide hormone with pressor and oxytocic activity, and has been synthesized. The decapeptide, angiotensin I ( X I I I ) , is enzymically cleaved to the octapeptide, angiotensin I I ( X I V ) , which is the active form of the hormone. Structure ( X I I I ) represents angiotensin I isolated from equine and hog plasma. Bovine plasma affords the related valine-5-angiotensin I .
< Angiotensin I ( X I I I ) • Asp · Arg · Val · T y r · lieu · His · Pro · Phe · His · Leu
1 2 3 4 5 6 7 8 9 10
< Angiotensin I I ( X I V ) >
Bumpus et al. (SOI), in a paper on the relationship of structure to pressor and oxytocic activity of isoleucine-angiotensin and various analogues, have presented a summary of this field together with the pertinent refer
ences. The proceedings of a recent symposium on angiotensin have been published (301a).
With one exception all of the analogues synthesized to date exhibit reduced pressor and oxytocic activity. The octapeptide, alanines-angio
tensin ( X V ) , in which alanine replaces proline, shows depressor activity (302).
Asp · Arg · Val · T y r · lieu · His · Ala · Phe ( X V )
The structure elucidation and synthesis of other polypeptide hormones, such as bradykinin (303, 303a), has led to the synthesis of analogues. The generalization still holds that such analogues, if they are at all active, ex
hibit hormone-like activity to a reduced degree rather than inhibitory activity. However, it is reasonable to expect that extension of this approach will lead to interesting and useful metabolic antagonists of the polypeptide hormones, which will be of importance in elucidating the mechanisms of hormone action and control, as well as in the therapy of hormonal disorders.
2. CHEMICALLY UNDEFINED INHIBITORS
Some work has been done on polypeptide hormone inhibitors that have not been completely purified and characterized.
a. Inhibitors of Thyrotropic Hormone (TSH). Sonenberg and Money (804) have reported that acetylated pituitary extracts inhibit the thyro
tropic but not the gonadotropic activity of the native preparations. It is likely that the inhibitor is an acetylated form of thyrotropic hormone (TSH). These investigators have suggested that if there is a limited num
ber of binding sites in the thyroid available for unmodified TSH, the inhibitory acetylated thyrotropin may inhibit TSH by competition for these binding sites. However, since TSH is not yet available in pure form, the possibility cannot be excluded at present that the inhibitor derives from some other component initially present in the pituitary preparation, and might be acetylated gonadotropin or another acetylated protein.
b. Inhibitors of Relaxin. Preparation of relaxin, isolated from the ovaries of pregnant sows, have been shown to block the formation of decidua in the uteri of ovariectomized, pseudopregnant rats treated with progesterone (305). When an excess of inactivated relaxin, chemically modified with thioglycollate or mercaptoethanol, is administered simultaneously with native relaxin, this property of relaxin, but not the symphyseal relaxation in guinea pigs, is nearly completely abolished in a reversible manner (306).
Since pure relaxin was not available and since similar results have been obtained with chemically reduced ovarian preparations of initially low relaxin content, it should be noted that the observed inhibition of relaxin may be a property of a chemically modified substance other than relaxin.
c. Inhibitors of Vasopressin. Inhibition in the rat of the pressor activity of arginine-vasopressin by preparations of the latter that have been treated with acetic anhydride has been observed (289).
B. Inhibitors of Hormone-Inactivating Systems
Another class of polypeptide hormone analogues with inhibitory proper
ties comprises chemically altered derivatives that, paradoxically, poten
tiate the activity of the native hormone by interfering with the enzymic system which normally degrades or inactivates the hormone.
1. INHIBITORS OF OXYTOCIN INACTIVATION
The first example of this phenomenon is the observation of Berânkovâ et al. (807) that the inactivation of oxytocin by "oxytocinase" could be inhibited in vitro by simple peptides, corresponding to structural fragments of the hormone, which may act as specific enzyme inhibitors by virtue of serving as competitive substrates. One such inhibitory peptide is cystinyl-bis(prolylleucylglycinamide).
2. INHIBITORS OF CORTICOTROPIN ( A C T H ) INACTIVATION
Another such inhibitor is an oxidized derivative of corticotropin (ACTH).
Cohen and Frieden (808) have found that the addition of this inhibitor, inactivated by oxidation with potassium persulfate, potentiates the ac
tivity of native A C T H , apparently by a similar mechanism. This analogue, which possessed no hormonal activity itself, was effective in potentiating the biological activity of A C T H as much as tenfold, as measured by the A
4
-3-ketosteroid secretion of mouse adrenocortical tumor slices in vitro (809). The chemical structure of the oxidized derivative is unknown, ex
cept that the polypeptide chain appears to be intact and that the ultra
violet absorption spectrum is different. Oxidation products of other poly
peptide hormones, glucagon and insulin, did not exhibit this inhibition of the ACTH-inactivating system.