The multi-specific human 17 beta-hydroxysteroid dehydrogenase type 7: Non-competitive inhibitors can target different catalyses to facilitate breast cancer treatment.

Human 17ß-hydroxysteroid dehydrogenase type 7 (17ß-HSD7), a special multifunctional enzyme, activates the estrogen estrone while inactivating the potent androgen dihydrotestosterone. Thus, this enzyme has become an ideal target for hormone-dependent breast cancer treatment, as its inhibition leads to estradiol reduction and dihydrotestosterone restoration. However, a particular concern has arisen related to an additional role in cholesterol biosynthesis, as inhibition of the enzyme may lead to undesirable side effects. Our findings demonstrate that the available enzyme inhibitors are non-competitive. Among these, many such as INH81, are specific toward sex-hormone conversion, whereas others represented by 4-bromo-ethynylestradiol, are more specific for zymosterone reduction occurring during cholesterol biosynthesis. The binding of non-competitive inhibitors does not affect the substrate binding on the enzyme. This is the first demonstration of non-competitive inhibitors acting selectively on different catalyses, thereby facilitating inhibitor uses for breast cancer treatment. We aim to quickly communicate the novel results.

Novel Coronavirus Polymerase and Nucleotidyl-Transferase Structures: Potential to Target New Outbreaks.

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.

The dual sex hormone specificity for human reductive 17ß-hydroxysteroid dehydrogenase type 7: Synergistic function in estrogen and androgen control.

Human 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 and 7 catalyze the final step of estrogen activation and the first step in androgen inactivation. It has been shown in breast cancer cells that DHT has a suppression effect on cell proliferation, counteracting the estrogen growth effect. However, the exact kinetic function of 17ß-HSD7 in steroidogenesis was not determined. Here we report the steady-state kinetics and binding study for 17ß-HSD7 with estrone or DHT as substrates and NADPH as cofactor. 17ß-HSD7 has been overexpressed in E. coli and purified. For both substrates, kinetics of 17ß-HSD7 demonstrates positive cooperativity. The K0.5 value is 5.2 ± 0.4 µM and 14.4 ± 0.8 µM and the kcat is 0.0063 ± 0.0003 s-1 and 0.0153 ± 0.0007 s-1 for the reduction of E1 and DHT, respectively. The binding study shows a similar affinity with a dissociation constant of 5.2 ± 0.5 µM and 11 ± 1 µM for E1 and DHT, respectively. Our kinetic and binding results reveal a positive cooperativity for 17ß-HSD7 to both the E1 and DHT with a similar affinity, while 17ß-HSD1 demonstrated a significantly higher affinity toward E1 than DHT, but with a strong E1 substrate inhibition. These results strongly support that the inhibition of 17ß-HSD7 constitutes the basis of breast cancer cell proliferation decreasing that led to the shrinkage of xenograft ER + breast tumor mice model.

Substrate inhibition of 17ß-HSD1 in living cells and regulation of 17ß-HSD7 by 17ß-HSD1 knockdown.

This study addresses first the role of human 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) in breast cancer (BC) cells. The enzyme has a high estrone-activating activity that is subject to strong substrate inhibition as shown by enzyme kinetics at the molecular level. We used BC cells to verify this phenomenon in living cells: estrone concentration increase did reduce the reaction with 0.025 to 4µM substrate. Moreover, 5α-dihydrotestosterone (DHT) demonstrated some inhibition of estrogen activation at both the molecular and cellular levels. The presence of DHT did not change the tendency toward substrate inhibition for estrone conversion, but shifted the inhibition toward higher substrate concentrations. Moreover, a binding study demonstrated that both DHT and dehydroepiandrosterone (DHEA) can be bound to the enzyme, thereby supporting the multi-specificity of 17ß-HSD1. We then followed the concentrations of estradiol and performed q-RT-PCR measurements of reductive 17ß-HSDs after 17ß-HSD1 inhibition. The estradiol decrease by the 17ß-HSD1 inhibition was demonstrated lending support to this observation. Knockdown and inhibition of 17ß-HSD1 produced reduction in estradiol levels and the down-regulation of another reductive enzyme 17ß-HSD7, thus "amplifying" the reduction of estradiol by the 17ß-HSD1 modulation itself. The critical positioning of 17ß-HSD7 in sex-hormone-regulation as well as the mutual regulation of steroid enzymes via estradiol in BC, are clearly demonstrated. Our study demonstrates that fundamental enzymological mechanisms are relevant in living cells. Moreover, further enzyme study in cells is merited to advance biological and medical research. We also demonstrated the central role of 17ß-HSD7 in sex-hormone conversion and regulation, supporting it as a novel target for estrogen-dependent (ER+) BC.

Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.

Impact of structural modifications at positions 13, 16 and 17 of 16ß-(m-carbamoylbenzyl)-estradiol on 17ß-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity.

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16ß-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17ß-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1µM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1µM, not at all at 0.1µM, and showed an interesting inhibitory potency on 17ß-HSD1 (90, 59 and 22%, at 10, 1 and 0.1µM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17ß-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies.

Synergistic control of sex hormones by 17ß-HSD type 7: a novel target for estrogen-dependent breast cancer.

17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17ß-HSD type 7 (17ß-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17ß-HSD7 in breast carcinoma. Inhibition of 17ß-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17ß-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17ß-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.