Lawrence B. Hendry

Emerging diversities in the mechanism of action of steroid hormones

The classical genomic action of steroid hormones acting through intracellular receptors is well recognized. Within this concept of action, questions regarding the ultimate fate of the hormone and lack of a tight correlation between tissue uptake and biological activity with receptor binding remain unanswered. Evidence has accumulated that steroid hormones can exert non-classical action that is characterized by rapid effect of short duration. In most of these cases, the hormone effects occurs at the membrane level and is not associated with entry into the cell. The possible mechanisms for these non-classical actions are: (a) changes in membrane fluidity; (b) steroid hormone acting on receptors on plasma membranes; (c) steroid hormones regulating GABAA receptors on plasma membranes; and (d) activation of steroid receptors by factors such as EGF, IGF-1 and dopamine. Data have also been obtained indicating that receptor-mediated insertion of steroid hormones into DNA may take place with the steroid acting as a transcription factor. These new proposed mechanism of action of steroid hormones should not be viewed as a challenge to the classical mechanism. These diverse modes of action provide for an integrated action of hormones which may be rapid and of short duration or prolonged to address the physiological needs of the individual.

Diverse modes of action of progesterone and its metabolites

Progesterone and its metabolites have a variety of diverse effects in the brain, uterus, smooth muscle, sperm and the oocyte. The effects include changes in electrophysiological excitability, induction of anesthesia, regulation of gonadotropin secretion, regulation of estrogen receptors, modulation of uterine contractility and induction of acrosome reaction and oocyte maturation. The latency of the effects vary from several seconds to several hours. Thus, it is not surprising that multiple mechanisms of action are involved. The classical mechanism of steroid hormone action of intracellular receptor binding has been supplemented by the possibility of the steroid acting as a transcription factor after the binding of the receptor protein to DNA. Other mechanisms include influence of the steroids on membrane fluidity and acting through other cell signalling systems, membrane receptors and GABA(A) receptors. Of particular interest are multiple mechanisms for the same types of action. For example the effect of progesterone on gonadotropin release is largely exerted via the classical intracellular receptor as well as membrane receptors, whereas 3(alpha),5(alpha)-tetrahydroprogesterone-induced LH release occurs via the GABA(A) receptor system. The inhibition of uterine contractility by progesterone is regulated by progesterone receptors while the action of 3(alpha),5(alpha)-tetrahydroprogesterone on uterine contractility is regulated by GABA(A) receptors. The regulation of the differences in the pattern of progesterone effects on estrogen receptor dynamics in the anterior pituitary and the uterus in the same animal are also of considerable interest.

Structure — activity relationships of some unique estrogens related to estradiol are predicted by fit into dna

The estrogenic activity of 11 beta-acetoxy estradiol, 11 beta-hydroxy estradiol, 11 alpha-hydroxy estradiol and 9 beta-estradiol was compared to estradiol using the restoration of uterine weight and prevention of LH rise in immature ovariectomized rats as endpoints of the assay. There was a good correlation between results using the two methods and estrogenic activity was found to be in the following order: 11 beta-acetoxy estradiol greater than estradiol greater than 9 beta-estradiol greater than 11 beta-hydroxy estradiol greater than 11 alpha-hydroxy estradiol. The biological activities of these compounds could be explained on the basis of stereochemical complementarity to the structure of DNA.

Drug design with a new type of molecular modeling based on stereochemical complementarity to gene structure.

Why certain chemical structures and not others are present in nature has been a recurring question raised by scientists since the first organic natural products were characterized. Of equal interest has been elucidating what structural features within any given class of organic molecules are responsible for biological activity. Historically, the lack of satisfactory answers to both questions has relegated the development of biologically active molecules either to serendipity or to exhaustive synthesis and biological testing of large numbers of compounds. This frustration is particularly evident in the pharmaceutical industry where the development of drug agonists and antagonists is often time consuming, tedious and expensive. Fortunately, this picture is beginning to change as more information is derived from modern molecular modeling techniques including characterization of the active sites in enzymes and the ligand binding sites in receptors. Over the past 15 years another approach has emerged based upon a series of discoveries made in our laboratories with molecular models. Namely, many biologically active small molecules have been found to possess complementary stereochemical relationships with gene structure. These relationships have proven useful in understanding constraints imposed by nature on the structures of small molecules and in correlating structure with activity among certain classes of compounds. Recently, computer graphics and energy calculations have confirmed salient observations lending credence to what promises to be a powerful and rapidly evolving technology for designing new safe and effective drugs.

Hypoglycemia caused by selegiline, an antiparkinsonian drug: can such side effects be predicted?

Treatment with selegiline produced profound hypoglycemia in a 70‐year‐old man with Parkinsons disease. The hypoglycemia was accompanied by hyperinsulinemia and persisted for 1 week after selegiline was discontinued. Although this side effect of antidepressant monoamine oxidase inhibitors was well documented in 1959–1968 publications, it was not known to the manufacturer of selegiline. Effects of drugs on glucose metabolism may be predictable through a novel molecular modeling technique developed in our laboratories, which shows that glucose exhibits stereochemical complementarity to a specific site in partially unwound DNA. Selegiline and other molecules affecting glucose metabolism fit into the same DNA base sequence. It therefore should be possible to employ this technique to identify pharmaceutical agents that possess hypoglycemic or hyperglycemic effects in vivo.

Apparent stereochemical complementarity of estrogens and helical cavities between DNA base pairs: Implications for the mechanism of action of steroids

The shape of the space occupied by a model of the estrogenic steroid hormone estradiol-17 beta conforms closely to a helical cavity between neighboring base pairs in partially coiled B-DNA. The orientation of estradiol-17 beta when fitted into DNA allows stereochemically complementary hydrogen bonding of both the 3- and 17 beta-hydroxyl groups to phosphate oxygens of the deoxyribose-phosphate backbone on adjacent strands. Changes in the chirality (handedness) of the steroid skeleton or in the absolute stereochemistry of hydrogen bonding groups prevent formation of complementary fits in the DNA. Synthetic estrogens can also adopt conformations which are stereochemically complementary to the cavities between base pairs. The complementary relationships between active estrogens and nucleic acids may be related to constraints on the evolution of the structure and the biological function of steroids.

Inhibition of estrogen stimulated mitogenesis by 3-phenylacetylamino-2,6-piperidinedione and its Para-hydroxy analog

3-Phenylactetylamino-2,6-piperidinedione (A10) inhibited estradiol stimulated cell growth in the MCF-7 (E3) human breast tumor cell line in vivo and in vitro. While high concentrations of A10 were needed to inhibit cell proliferation (IC50 = 3 x 10(-3) M in vitro), the compound demonstrated little toxicity. The effect appeared specific since a hydrolysis product of A10, phenylacetylglutamine, demonstrated no growth inhibitory activity at similar concentrations in MCF-7 (E3) cells in vitro. A computer designed analog, p-hydroxy A10, was more potent than A10 in inhibiting activity in MCF-7 (E3) cells in vitro. The IC50 for p-hydroxy A10 was 7 x 10(-6) M which was comparable to that of the antiestrogen, tamoxifen (IC50 1 x 10(-7) M). All three compounds caused a decline in estrogen receptor levels in a dose-dependent fashion. A10 also inhibited estradiol induction of progesterone receptors. Examination of protein kinase activity following an acute exposure to a 10(-11) M growth stimulatory dose of estradiol revealed a 168% increase in protein kinase activity over that of untreated control cells. A10 in a dose-responsive fashion inhibited the estradiol stimulated increase in protein kinase activity. The protein kinase activity was also inhibited by p-hydroxy A10. These activities of A10 and p-hydroxy A10 coupled with the low toxicity and novelty of the basic A10 structure provide an exciting possibility of developing a new class of clinically useful antineoplastic drugs with minimal side effects.

Actions of an endogenous antitumorigenic agent on mammary tumor development and modeling analysis of its capacity for interacting with DNA

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione) is an agent derived from human urine which is remarkable for its antineoplastic activity and lack of toxicity. This study deals with the discovery of a hormonal component to the action of A10 on rodent tumor formation and approaches to the delineation of its mechanism of action. Oral administration of A10 dramatically delays onset of spontaneous mammary tumor incidence in female and orchidectomized male C3H+ mice. The action in the male qualitatively mimics that of androgens. In the rat, A10 ingestion is very effective in preventing carcinogen-induced mammary tumors, but does not cause regression of pre-established tumors. It selectively blocks the occurrence of the estrogen-sensitive subpopulation of tumors, acting in a fashion completely analogous to that of tamoxifen; in contrast to tamoxifen, however, A10 has no measurable affinity for the estrogen receptor. Modeling studies demonstrate a stereoselective capacity for interaction between A10 and specific deoxyribonucleotide base pair sequences. The potential affinity is lower than that for classical intercalating antitumor agents, and the stereospecificity differs from that which we have previously established for estrogens. We offer an interpretation of the data in terms of direct effect of A10 at the genomic level to alter the cellular responsiveness to steroid hormones.

Antiestrogenic piperidinediones designed prospectively using computer graphics and energy calculations of DNA-ligand complexes

Drug design technology based upon DNA stereochemistry and now supplemented by computer modeling was used to design a novel compound to inhibit estrogen-induced tumor cell growth. A known compound 3-phenylacetylamino-2,6-piperidinedione (PP) was accommodated in partially unwound DNA in a manner consistent with criteria for antiestrogens. Examination of the PP-DNA complex revealed that substitution of a hydroxyl group at the para position (p-OH-PP) would provide a stereospecific hydrogen bond and a substantial increase in fit as assessed by energy calculations. The antiestrogen tamoxifen could also be accomodated within the site; analogous substitution of a hydroxyl at the 4 position resulted in a better fitting molecule. 4-Hydroxytamoxifen is a more potent antiestrogen than tamoxifen. Synthesis and subsequent evaluation of p-OH-PP as an inhibitor of estrogen stimulated MCF-7 (E3) human breast cancer cell growth demonstrated that p-OH-PP was more active than both PP and its hydrolysis product phenylacetylglutamine. As predicted, the order of fit into DNA correlated with the relative ability to inhibit estrogen-induced growth of tumor cells suggesting that the evolving drug design technology will be valuable in developing new drugs for breast cancer.

Stereochemical complementarity of progesterone, RU486 and cavities between base pairs in partially unwound double stranded DNA assessed by computer modeling and energy calculations

Computer modeling was used to examine the relative fit of progesterone and RU486 in cavities constructed between base pairs in double stranded DNA. Progesterone was capable of forming two stereospecific hydrogen bonds between the carbonyl groups and protonated phosphate groups on adjacent strands. Favorable van der Waals and electrostatic energies were exhibited upon insertion of progesterone into DNA indicating an excellent fit. While RU486 could be accommodated between the base pairs and formed hydrogen bonds, there was a high van der Waals energy in the resulting complex. When the complexes were subjected to energy minimization, the conformation of the DNA was significantly altered in the RU486-DNA complex but not in the progesterone-DNA complex. No mechanistic interpretation of these results is proffered; however, such information may have evolutionary significance and could prove useful in designing new progesterone agonists and antagonists.