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SARS-CoV-2 exploits the homotrimer transmembrane Spike glycoproteins (S protein) during host cell invasion. Omicron, delta, and prototype SARS-CoV-2 receptor-binding domain show similar binding strength to hACE2 (Angiotensin-Converting Enzyme 2). Here we utilized multi-ligand virtual screening to identify small molecule inhibitors for their efficacy against SARS-CoV-2 virus using quantum Docking, pseudovirus ACE2 Inhibition TR-FRET Assay Screening, and Molecular Dynamic simulations (MDS). 350-thousand compounds were screened against the macrodomain of non-structural protein 3 of SARS-CoV-2. Using TR-FRET Assay, we filtered out two of 10 compounds that had no reported activity inin-vitroscreen against Spike S1: ACE2 binding assay. Percentage Inhibition at 30 µM was found to be 79% for “Compound F1877-0839” and 69% for “Compound F0470-0003”. This first of its kind study identified “FILLY” pocket in macrodomains. Our 200 ns MDS revealed stable binding poses of both leads. They can be used for further development of preclinical candidates.Abstract ImageIn BriefIqbal et al., described a deep learning guided drug discovery, efficacy against SARS-CoV-2 Spike inhibitors: using experimental pseudovirus ACE2 Inhibition TR-FRET Assay. Our molecular dynamic simulation results were next validated a posteriori against the corresponding experimental data of identified leads with 80 percent inhibition. Moreover, this study is first of kind to identify “FILLY” pocket in macrodomains.HighlightsExperimental pseudovirus ACE2 Inhibition TR-FRET Assay and HTS lead to identification of two potential clinical leads.Conformational Dynamics analysis reveal the structural stability of complexes throughout 200 ns molecular dynamic simulations.Unveiling of the impact surface charge on the Variant of ConcernsDetection of conformational changes within ACE2/RBD complexWe identified the FILLY pocket in the SARS viruses.

Human 17β-hydroxysteroid dehydrogenase type 7 (17β-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.

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17β-HSD members, 17β-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17β-HSD1 in complex with estrone (E1) and cofactor analog NADP+ , demonstrating critical enzyme-substrate-cofactor interactions. These complexes revealed a reversely bound E1 in 17β-HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His221 is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead-end complex, which exists widely in NADP(H)-preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17β-HSD1 through the formation of a dead-end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17β-HSD1 based on the dead-end inhibition model for efficiently targeting EDDs. DATABASES: Coordinates and structure factors of 17β-HSD1-E1 and 17β-HSD1-E1-NADP+ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively. ENZYMES: 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) EC 1.1.1.62.

Cyclin-dependent kinases (CDKs) are historic therapeutic targets implicated in tumorigenic events due to their critical involvement in the cell cycle phase. However, selectivity has proven to be a bottleneck, causing repeated failures. Previously, we reported CR6-interacting factor 1 (CRIF1), acting as a cell cycle negative regulator through interaction with CDK2. In the current report, we identified the CRIF1-CDK2 interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on CRIF1 using 40 678 drug-like compounds. These compounds were tested by cell proliferation assay, and four of these molecules were found to selectively inhibit the proliferation of osteosarcoma (OS) cell lines, but do not affect normal bone mesenchymal stem cells (BMSC). A binding study reveals significant affinities of the inhibitors on CRIF1. More importantly, treatment of the OS cells with a combination of ionizing radiation (IR) and the best-performing inhibitors remarkably increased IR inhibition potential from 19.9% to 59.6%. This occurred by selectively promoting G2/M arrest and apoptosis related to CDK2 overactivation in OS cells but not in BMSC and was supported by significant CDK2 phosphorylation modifications. Knocking down of CRIF1 by siRNA treatment showed similar effects to the interface inhibitors. Together we substantiate the identification of novel lead molecules, which may provide a new treatment to overcome selectivity issues and enhance the radiosensitivity of tumor cells, opening a conceptually novel strategy of CDK-targeting for different cancer types.

Optical memory based on the electromagnetically induced transparency (EIT) in a double-$$\Lambda$$ Λ atomic system provides a convenient way to convert the frequency, bandwidth or polarization of an optical pulse by storing it in one $$\Lambda$$ Λ channel and retrieving it from another. This memory-based optical converter can be used to bridge the quantum nodes which have different physical properties in a quantum network. However, in real atoms, each energy level usually contains degenerate Zeeman states, which may lead to additional energy loss, as has been discussed in our recent theoretical paper (Tsai et al. in Phys. Rev. A 100, 063843). Here, we present an experimental study on the efficiency variation in the EIT-memory-based optical polarization conversion in cold cesium atoms under Zeeman-state optical pumping. The experimental results support the theoretical predictions. Our study provides quantitative knowledge and physical insight useful for practical implementation of an EIT-memory-based optical converter.

Optical memory based on the electromagnetically induced transparency (EIT) in a double-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda$$\end{document}Λ atomic system provides a convenient way to convert the frequency, bandwidth or polarization of an optical pulse by storing it in one \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda$$\end{document}Λ channel and retrieving it from another. This memory-based optical converter can be used to bridge the quantum nodes which have different physical properties in a quantum network. However, in real atoms, each energy level usually contains degenerate Zeeman states, which may lead to additional energy loss, as has been discussed in our recent theoretical paper (Tsai et al. in Phys. Rev. A 100, 063843). Here, we present an experimental study on the efficiency variation in the EIT-memory-based optical polarization conversion in cold cesium atoms under Zeeman-state optical pumping. The experimental results support the theoretical predictions. Our study provides quantitative knowledge and physical insight useful for practical implementation of an EIT-memory-based optical converter.

We present a theoretical study on the efficiency variation of coherent light conversion based on optical memories using the electromagnetically induced transparency (EIT) protocol in an atomic system with degenerate Zeeman states. Based on the Maxwell-Bloch equation, we obtain an approximate analytic solution for the converted light pulses which clarifies that two major factors affecting the efficiency of the converted pulses. The first one is the finite bandwidth effect of the pulses and the difference in the delay-bandwidth product of the writing and reading channel due to the difference in the transition dipole moment. The second one is the mismatch between the stored ground-state coherence and the ratio of the Clebsch-Gordan coefficient for the probe and control transition in the reading channel which results in a non-adiabatic energy loss. To correspond to real experimental conditions, we also perform a numerical calculation of the variation in conversion efficiency versus the Zeeman population distribution under the Zeeman-state optical pumping in storing a $\sigma^{+}$-polarized pulse and retrieving with $\sigma^{-}$ polarization in cesium atoms. Our work provides essential physical insights and quantitative knowledge for the development of a coherent optical converter based on EIT-memory., Comment: 12 pages, 10 figures

As the field of superconducting quantum computing approaches maturity, optimization of single-device performance is proving to be a promising avenue toward large-scale quantum computers. However, this optimization is possible only if performance metrics can be accurately compared among measurements, devices, and laboratories. Currently, such comparisons are inaccurate or impossible due to understudied errors from a plethora of sources. In this Perspective, we outline the current state of error analysis for qubits and resonators in superconducting quantum circuits and discuss what future investigations are required before superconducting quantum device optimization can be realized.

Superconducting circuit testing and materials loss characterization requires robust and reliable methods for the extraction of internal and coupling quality factors of microwave resonators. A common method, imposed by limitations on the device design or experimental configuration, is the single-port reflection geometry, i.e. reflection-mode. However, impedance mismatches in cryogenic systems must be accounted for through calibration of the measurement chain while it is at low temperatures. In this paper, we demonstrate a data-based, single-port calibration using commercial microwave standards and a vector network analyzer (VNA) with samples at millikelvin temperature in a dilution refrigerator, making this method useful for measurements of quantum phenomena. Finally, we cross reference our data-based, single-port calibration and reflection measurement with over-coupled 2D- and 3D-resonators against well established two-port techniques corroborating the validity of our method.

Highlights • Antivirals targeting RdRp, proteases and Spike protein from SARS-CoV-2. • Drug searching combining structural biology and virology. • Drug repurposing, HTS and virtual inhibitor screening to find antivirals., Graphical abstract Highlights • Antivirals are targeting RdRp, proteases and Spike protein from SARS-CoV-2. • Drug searching combining structural biology and virology. • Drug repurposing, HTS and virtual inhibitor screening to find antivirals., The global spread of COVID-19 constitutes the most dangerous pandemic to emerge during the last one hundred years. About seventy-nine million infections and more than 1.7 million death have been reported to date, along with destruction of the global economy. With the uncertainty evolved by alarming level of genome mutations, coupled with likelihood of generating only a short lived immune response by the vaccine injections, the identification of antiviral drugs for direct therapy is the need of the hour. Strategies to inhibit virus infection and replication focus on targets such as the spike protein and non-structural proteins including the highly conserved RNA-dependent-RNA-polymerase, nucleotidyl-transferases, main protease and papain-like proteases. There is also an indirect option to target the host cell recognition systems such as angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2, host cell expressed CD147, and the host furin. A drug search strategy consensus in tandem with analysis of currently available information is extremely important for the rapid identification of anti-viral. An unprecedented display of cooperation among the scientific community regarding SARS-CoV-2 research has resulted in the accumulation of an enormous amount of literature that requires curation. Drug repurposing and drug combinations have drawn tremendous attention for rapid therapeutic application, while high throughput screening and virtual searches support de novo drug identification. Here, we examine how certain approved drugs targeting different viruses can play a role in combating this new virus and analyze how they demonstrate efficacy under clinical assessment. Suggestions on repurposing and de novo strategies are proposed to facilitate the fight against the COVID-19 pandemic.

This study addresses first the role of human 17β-hydroxysteroid dehydrogenase type 1 (17β-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 17β-HSD1. We then followed the concentrations of estradiol and performed q-RT-PCR measurements of reductive 17β-HSDs after 17β-HSD1 inhibition. The estradiol decrease by the 17β-HSD1 inhibition was demonstrated lending support to this observation. Knockdown and inhibition of 17β-HSD1 produced reduction in estradiol levels and the down-regulation of another reductive enzyme 17β-HSD7, thus "amplifying" the reduction of estradiol by the 17β-HSD1 modulation itself. The critical positioning of 17β-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 17β-HSD7 in sex-hormone conversion and regulation, supporting it as a novel target for estrogen-dependent (ER+) BC.

Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barre syndrome in adults. At present there is limited understanding of the infectious mechanism and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Nonstructural protein 5 (NS5) is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp and by molecular docking and phylogeny analysis we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. This article is protected by copyright. All rights reserved.

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p-aminobenzamidine-modified affinity chromatography. The affinity medium was synthesized by using p-aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant Kd and theoretical maximum adsorption Qmax of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high-performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.

Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28 341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 μM respectively, identifying lead compounds for anti-ZIKV drug development.

The data presented here are related to the research article entitled “Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1” (J.A. Aka, E.L. Calvo, S.X. Lin, 2016) [1]. We evaluated the effect of the steroidal enzyme 17β-HSD1 and its product, the estrogenic hormone 17-beta-estradiol (E2), on gene transcription profile of breast cancer cells. RNA interference technique was used to knock down the 17β-HSD1 gene (HSD17B1) in the hormone-dependent breast cancer cell line T47D in steroid-deprived medium. Transfected cells were subsequently treated with E2, and microarray analyses (with three contrasts) were used to investigate (i) the effect of 17β-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition, (ii) the effect of E2 on breast cancer gene expression and (iii) if E2 affects gene regulation by 17β-HSD1. Functional enrichments of the differentially expressed genes were assessed using Ingenuity Pathway Analysis (IPA). Here, we showed data on 140 genes that are induced or repressed 1.5 time or higher (p < 0.05) in the HSD17B1-silenced and E2-treated T47D cells revealed by microarray analysis, and presented the 14 functional terms found in the cancer and in the cell death and survival categories revealed by the IPA biological function analysis. Data on IPA Canonical Pathway and network analyses is also presented. Further discussion on gene regulation by 17β-HSD1 and E2 is provided in the accompanying publication [1].

Breast cancer (BC) and prostate cancer (PC) are the second most common malignant tumors in women and men in western countries, respectively. The risks of death are 14% for BC and 9% for PC. Abnormal estrogen and androgen levels are related to carcinogenesis of the breast and prostate. Estradiol stimulates cancer development in BC. The effect of estrogen on PC is concentration-dependent, and estrogen can regulate androgen production, further affecting PC. Estrogen can also increase the risk of androgen-induced PC. Androgen has dual effects on BC via different metabolic pathways, and the role of the androgen receptor (AR) in BC also depends on cell subtype and downstream target genes. Androgen and AR can stimulate both primary PC and castration-resistant PC. Understanding the mechanisms of the effects of estrogen and androgen on BC and PC may help us to improve curative BC and PC treatment strategies.

In spite of the significant progress of estrogen-dependent breast cancer (BC) treatment, aromatase inhibitor resistance is a major problem limiting the clinical benefit of this frontier endocrine-therapy. The aim of this study was to determine the differential expression of steroid-converting enzymes between tumor and adjacent normal tissues, as well as their correlation in modulating intratumoral steroid-hormone levels in post-menopausal estrogen-dependent BC. RNA sequencing dataset (n = 1097) of The-Cancer-Genome-Atlas (Breast Invasive Carcinoma) retrieved through the data portal of Genomic Data Commons was used for differential expressions and expression correlation analyses by Mann-Whitney U and Spearman's rank test, respectively. The results showed significant up-regulation of 17β-HSD7 (2.50-fold, p 0.0001) in BC, supporting its effect in sex-hormone control. Besides, suppression of 11β-HSD1 expression (-8.29-fold, p 0.0001) and elevation of 11β-HSD2 expression (2.04-fold, p 0.0001) provide a low glucocorticoid environment diminishing BC anti-proliferation. Furthermore, 3α-HSDs were down-regulated (-1.59-fold, p 0.01; -8.18-fold, p 0.0001; -33.96-fold, p 0.0001; -31.85-fold, p 0.0001 for type 1-4, respectively), while 5α-reductases were up-regulated (1.41-fold, p 0.0001; 2.85-fold, p 0.0001; 1.70-fold, p 0.0001 for type 1-3, respectively) in BC, reducing cell proliferation suppressers 4-pregnenes, increasing cell proliferation stimulators 5α-pregnanes. Expression analysis indicates significant correlations between 11β-HSD1 with 3α-HSD4 (r = 0.605, p 0.0001) and 3α-HSD3 (r = 0.537, p 0.0001). Significant expression correlations between 3α-HSDs were also observed. Our results systematically present the regulation of steroid-converting enzymes and their roles in modulating the intratumoral steroid-hormone levels in BC with a vivid 3D-schema, supporting novel therapy targeting the reductive 17β-HSD7 and proposing a new combined therapy targeting 11β-HSD2 and 17β-HSD7.

We report loading of laser-cooled caesium atoms into a hollow-core photonic-bandgap fiber and confining the atoms in the fiber’s 7μm diameter core with a red-detuned dipole trap. In this system, the atom-photon interaction probability is in the range of 0.5% and optical depths exceeding 100 can be achieved. We discuss the outlooks for photon storage and nonlinear optics at low light levels, such as cross-phase modulation and single-photon wavelength conversion, in this system.

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17β-HSD members, 17β-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17β-HSD1 in complex with estrone (E1) and cofactor analog NADP

At the late 1940s, 17β-HSD1 was discovered as the first member of the 17β-HSD family with its gene cloned. The three-dimensional structure of human 17β-HSD1 is the first example of any human steroid converting enzyme. The human enzyme's structure and biological function have thus been studied extensively in the last two decades. In humans, the enzyme is expressed in placenta, ovary, endometrium and breast. The high activity of estrogen activation provides the basis of 17β-HSD1's implication in estrogen-dependent diseases, such as breast cancer, endometriosis and non-small cell lung carcinomas. Its dual function in estrogen activation and androgen inactivation has been revealed in molecular and breast cancer cell levels, significantly stimulating the proliferation of such cells. The enzyme's overexpression in breast cancer was demonstrated by clinical samples. Inhibition of human 17β-HSD1 led to xenograft tumor shrinkage. Unfortunately, through decades of studies, there is still no drug using the enzyme's inhibitors available. This is due to the difficulty to get rid of the estrogenic activity of its inhibitors, which are mostly estrogen analogues. New non-steroid inhibitors for the enzyme provide new hope for non-estrogenic inhibitors of the enzyme.

Following menopause virtually 100% of estrogens are synthesized in peripheral target tissues from precursor steroids of adrenal origin. These steroids are the unique source of sex steroids in these women. This positions some steroid metabolizing enzymes as primary targets for novel therapies for estrogen receptor-positive (ER+) breast cancer. However, previous research on the steroid-converting enzymes has been performed using their direct substrate as a hormone source, depending on the facility where studied and the robust signal obtained. These experiments may not always provide an accurate reflection of physiological and post-menopausal conditions. We suggest providing dehydroepiandrosterone (DHEA) as an intracrinological hormone source, and comparing the role of steroid-converting enzymes using DHEA and their direct substrates when an extensive mechanistic understanding is required. Here, we present a comparative study of these enzymes with the provision of DHEA and the direct substrates, estrone (E1) or dihydrotestosterone (DHT), or additional steroids as hormone sources, in breast cancer cells. Enzyme knockdown by respective specific siRNAs and observations on the resulting differences in biological function were carried out. Cell biology studies showed no difference in biological function for 17β-HSD1 and 17β-HSD7 when cultured with different steroid hormones: cell proliferation and estradiol levels decreased, whereas DHT accumulated; cyclinD1, PCNA, and pS2 were down-regulated after knocking down these two enzymes, although the quantitative results varied. However, culture medium supplementation was found to have a marked impact on the study of 3α-HSD3. We demonstrated that provision of different steroids as a substrate or hormone sources may promote modified biological effects: provision of DHEA is the preferred choice to mimic postmenopausal steroid metabolism in cell culture.

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.

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.

Androgen receptor (AR) signaling is essential for prostate cancer (PC) progression and treatment. Experiments have demonstrated that the intratumoral androgen levels are not affected by circulating androgen levels, but rather modulated by local steroid-converting enzyme activities. The expression modulation status of human steroid-converting enzymes and nuclear receptors are of great promise to identify novel therapeutic targets. Meta-analysis was performed with 9 cohorts (1093 specimens) from Gene Expression Omnibus, 16 cohorts (933 specimens) from Oncomine and the TCGA cohort (550 specimens). We found significant up regulation of 5α-reductase type 1 and type 3 in both primary and metastatic PC, together with the down regulation of AKR1C2 in primary PC, contributing to the high intratumoral DHT levels. The expression of AR in metastatic PC was up regulated, indicating the importance of AR signaling in the progression of this cancer. The down regulations of HSD11B1 and NR3C1 in primary and metastatic PC may diminish the anti-inflammation and anti-proliferation effects of glucocorticoids signaling. Furthermore, the decrease of progesterone receptor (PGR) expression in primary and metastatic PC was also observed, relieving the suppression effect of PGR on PC proliferation. The clinical evidences of the remarkable expression modulation of steroid-converting enzymes and receptors in PC may indicate novel combined treatment against this highly incident cancer.

Breast cancer is a major cause of cancer-related death for women in western countries. 17β-Hydroxysteroid dehydrogenases (17β-HSDs) play important roles in the last step of sex-hormone activation and the first step of sex-hormone inactivation. 17β-HSD2 is responsible for oxidizing the sex hormones. We used microarray technology to analyze the effect of 17β-HSD2 on the MCF-7 cell transcript profile after knocking down 17β-HSD2. Five hundred forty-two genes were regulated 1.5-fold or higher after treatment with 17β-HSD2 siRNA. Knocking down 17β-HSD2 interrupted nucleosome assembly. Pathway-Act-Network analysis showed that the MAPK and apoptosis signaling pathways were most regulated. In the gene-gene interaction network analysis, UGT2B15, which is involved in hormone metabolism, was the most regulated core gene. FOS, GREB1, and CXCL12 were the most regulated genes, and CXCL12 was related to tumor migration. Following 17β-HSD2 knock-down, the cell viability decreased to 75.9%. The S-phase percentage decreased by 19.4%, the Q2-phase percentage in cell apoptosis testing increased by 1.5 times, and cell migration decreased to 66.0%. These results were consistent with our gene chip analysis and indicated that 17β-HSD2 plays both hormone-dependent and hormone-independent enzymatic roles. In-depth investigations of this enzyme on the genomic level will help clarify its related molecular mechanisms.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16β-( m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16β-( m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17β-HSD1-2-MeO-CC-156-NADP

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-A resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.

Human 17β-hydroxysteroid dehydrogenase (17β-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 17β-HSD7 in steroidogenesis was not determined. Here we report the steady-state kinetics and binding study for 17β-HSD7 with estrone or DHT as substrates and NADPH as cofactor. 17β-HSD7 has been overexpressed in E. coli and purified. For both substrates, kinetics of 17β-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 17β-HSD7 to both the E1 and DHT with a similar affinity, while 17β-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 17β-HSD7 constitutes the basis of breast cancer cell proliferation decreasing that led to the shrinkage of xenograft ER + breast tumor mice model.

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA