In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future.

In vitro labeling of gonadal steroid hormone receptors in brain tissue sections.

Autoradiographic methods have been developed for measurement of gonadal steroid receptors in situ in brain tissue sections. Based on principles established previously for estrogen receptors in the rat brain using a 125I-labeled ligand, procedures have been developed for in vitro labeling of estrogen, androgen, and progestin receptors with commercially available tritiated ligands. Addition of protamine sulfate to the incubation buffer precipitates the receptors in situ in the tissue sections, allowing them to be detected autoradiographically after incubation with labeled steroid and subsequent washing to remove unbound and nonspecifically bound ligand. Occupied and unoccupied estrogen receptors can be measured selectively using appropriately modified incubation conditions. In the case of androgen and progestin receptors, unoccupied receptors are readily detected by in vitro labeling of tissue sections, but occupied receptors do not appear to label efficiently. Preliminary data suggest that these methods should be equally applicable to a variety of laboratory animals, including the rat, mouse, guinea pig, and monkey.

Characterization of a hepatic protein in nonhuman primates that binds mibolerone but not dihydrotestosterone or methyltrienolone.

During our studies of the hepatic androgen receptor in cynomolgus monkeys, tritiated mibolerone +/- a 200-fold excess of unlabeled mibolerone has been used to determine specific binding in cytosol. During time-course studies, high-capacity, unsaturable binding of [3H]mibolerone was noted after short-term incubations (4 h, 4 degrees C). When hepatic cytosol from male monkeys was incubated for 18 h at 4 degrees C, the high-capacity binding disappeared; saturable, high-affinity binding with characteristics consistent with the androgen receptor then could be identified. The characterization of [3H]mibolerone binding in molybdate-stabilized hepatic cytosol using sucrose density gradients and gel filtration yielded an unstable binding peak in addition to that of the androgen receptor. This lower molecular weight protein identified by gel filtration did not bind other androgens, including methyltrienolone, and did not have characteristics of other binding proteins that have been identified previously. This protein was not precipitated from 30% ammonium sulfate, which allowed it to be separated from the androgen receptor. Binding to this protein in ovariectomized female monkeys did not disappear with extended incubation at 4 degrees C, suggesting greater stability or a higher capacity. The function of this protein is not known, but both triamcinolone acetonide and contraceptive progestins appeared to displace tritiated mibolerone that was bound to it. This high-capacity binding of mibolerone interferes in the assessment of androgen receptor levels in these females unless it is eliminated. The synthetic androgen methyltrienolone does not bind to this protein and is a better choice for defining binding to the androgen receptor in these tissues.

Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol.

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.

Synthesis of high affinity fluorine-substituted ligands for the androgen receptor. Potential agents for imaging prostatic cancer by positron emission tomography.

We have prepared nine androgens substituted with fluorine at C-16 or C-20 to evaluate their potential, as positron emission tomographic (PET) imaging agents for prostatic cancer when labeled with the positron emitting radionuclide fluorine-18 (t1/2 = 110 min). These compounds represent members from the following classes of androgens: testosterone (T), 5 alpha-dihydrotestosterone (DHT), 7 alpha-methyl-19-nortestosterone (MNT), mibolerone (Mib), and metribolone (R1881). All of these compounds were prepared by functionalization of suitable androgen precursors, and the synthetic routes were developed to allow the introduction of fluorine by a fluoride ion displacement reaction late in the synthesis, as is required for the preparation of these compounds in fluorine-18 labeled form. We have also prepared four androgens in which the C-3 carbonyl or 17 beta-hydroxyl groups are replaced by fluorine. Most of the fluorine-substituted androgens show high affinity for the androgen receptor (AR), although fluorine substitution lowers their affinity by a small factor. None of the androgens where fluorine replaces oxygen functions at C-3 or C-17 have substantial affinity for AR. Derivatives of the natural androgens (T and DHT) as well as MNT have little affinity for other steroid hormone receptors (progesterone and mineralocorticoid receptors), whereas the Mib and R1881 derivatives have somewhat greater heterologous binding. With sex steroid binding protein, a human serum binding protein, the pattern of binding affinities is nearly the reverse, with derivatives of Mib, R1881 and MNT having low affinity, and DHT and T, high affinity. From these fluorine-substituted compounds, we can select several whose preparation in fluorine-18 labeled form for further tissue distribution studies is merited.

Comparative study of androgen binding in rat submandibular gland and prostate.

A comparative study of androgen binding in the cytosol and nuclear extract of the rat submandibular gland (SMG) and ventral prostate (VP) was investigated by using methyltrienolone (R1881), testosterone (T) and dihydrotestosterone (DHT) as the labeled ligands. The 3H-labeled steroid was incubated with the cytosol or nuclear KCl extract in a Tris-HCl buffer (pH 7.4) at 4 degrees C for 20 h. A sephacryl S-200 column chromatography showed a single binding peak in the nuclei and two critical peaks on the binding in the cytosol of SMG. The eluation patterns of SMG were similar to those of the nuclei and cytosol from rat VP. The binding proteins were recovered predominantly in the cytosol as 150-200 kDa mol wt and in the nuclei as 14-20 Kda mol wt. The cytosol and nuclei of rat SMG and VP have a common androgen binding subunit of the molecular weight of 14-20 kDa. R1881 bound to cytosolic protein with a higher affinity than DHT in SMG and VP. The amount of the R1881 specific binding in the cytosol of male SMG was similar to that of VP, and the DHT specific binding in the male SMG cytosol was about 1/3 of the binding in the cytosol of VP. R1881, DHT and T suppressed the 3H-R1881 binding completely in the cytosol of rat VP. Whereas, in the cytosol of rat SMG, the 3H-R1881 binding was slightly suppressed by R1881 at low concentrations and effectively suppressed by R1881 at higher concentrations. DHT and T were weak inhibitors to the 3H-R1881 binding in SMG, and E2 was almost ineffective on the binding in male SMG and VP. DHT bound specifically to the nuclear extract protein in VP. The DHT specific binding was not so significant as T and R1881 in the nuclei of SMG. However, the T binding in the male nuclei was strikingly augmented by simultaneous adding of T and DHT to the incubation medium. Such androgen binding augmentation was not shown in VP. This means that the nuclear 14-20 kDa protein in SMG have an ability to bound a large amount of androgen, whose character is far different from the androgen binding mechanisms in VP, and that transformed binding protein is androgen dependent in the nuclei of rat SMG. These results demonstrated that rat SMG and VP had the same of similar molecular weight of androgen binding protein in the cytosol and nuclei. However, the protein in the nuclei of SMG had a marked different character from VP.