Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

Isoprenoid flavonoids isolated from Sophora davidii and their activities induces apoptosis and autophagy in HT29 cells.

Four previously undescribed isoprenoid flavonoids (2-5) were isolated from Sophora davidii, along with five known analogues. The structures of the compounds were established through comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and absolute configurations determined by theoretical calculations, including ECD and NMR calculation. The cytotoxic effects of the isolated compounds on human HT29 colon cancer cells were evaluated using the MTT assay, compound 1 exhibited cytotoxicity against human HT29 colon cancer cells with an IC50 value of 8.39 ± 0.09 µM. Studies conducted with compound 1 in HT29 cells demonstrated that it may induce apoptosis and autophagy in HT29 by promoting the phosphorylation of P38 MAPK and inhibiting the phosphorylation of Erk MAPK.

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations -receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. Significance: The estrogen receptor-α (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

Discovery of novel covalent selective estrogen receptor degraders against endocrine-resistant breast cancer.

Endocrine-resistance remains a major challenge in estrogen receptor α positive (ERα+) breast cancer (BC) treatment and constitutively active somatic mutations in ERα are a common mechanism. There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrine-resistance. Given aberrant ERα activity, we herein report the identification of novel covalent selective estrogen receptor degraders (cSERDs) possessing the advantages of both covalent and degradation strategies. A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERα+ breast cancer cell lines including mutant ERα. Crystal structure of ERα‒29c complex alongside intact mass spectrometry revealed that 29c disrupted ERα protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11, thus enforcing a unique antagonist conformation and driving the ERα degradation. These significant effects of the cSERD on ERα homeostasis, unlike typical ERα degraders that occur directly via long side chains perturbing the morphology of H12, demonstrating a distinct mechanism of action (MoA). In vivo, 29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity. This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.

Stem Cell Therapy in Neonatal Hypoxic-Ischemic Encephalopathy and Cerebral Palsy: a Bibliometric Analysis and New Strategy.

The aim of this study was to identify related scientific outputs and emerging topics of stem cells in neonatal hypoxic-ischemic encephalopathy (NHIE) and cerebral palsy (CP) through bibliometrics and literature review. All relevant publications on stem cell therapy for NHIE and CP were screened from websites and analyzed research trends. VOSviewer and CiteSpace were applied to visualize and quantitatively analyze the published literature to provide objective presentation and prediction. In addition, the clinical trials, published articles, and projects of the National Natural Science Foundation of China associated with stem cell therapy for NHIE and CP were summarized. A total of 294 publications were associated with stem cell therapy for NHIE and CP. Most publications and citations came from the USA and China. Monash University and University Medical Center Utrecht produced the most publications. Pediatric research published the most studies on stem cell therapy for NHIE and CP. Heijnen C and Kavelaars A published the most articles. Cluster analyses show that current research trend is more inclined toward the repair mechanism and clinical translation of stem cell therapy for NHIE and CP. By summarizing various studies of stem cells in NHIE and CP, it is indicated that this research direction is a hot topic at present. Furthermore, organoid transplantation, as an emerging and new therapeutic approach, brings new hope for the treatment of NHIE and CP. This study comprehensively summarized and analyzed the research trend of global stem cell therapy for NHIE and CP. It has shown a marked increase in stem cell therapy for NHIE and CP research. In the future, more efforts will be made on exploring stem cell or organoid therapy for NHIE and CP and more valuable related mechanisms of action to achieve clinical translation as soon as possible.

Correlation between Polyphenol Contents and Antioxidant Activities in Different Echinacea Purpurea Varieties.

OBJECTIVE: Polyphenols are complex compounds containing multiple phenolic hydroxyl groups. They are widely distributed in plants and have antioxidant activities. Whether the antioxidant activities of the cultivated varieties of Echinacea are similar to or better than those of the wild ones and the relationship between the accumulation of polyphenols and their antioxidant activities are still not clear. METHODS: Folin-Ciocalteu method, high performance liquid chromatography (HPLC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric ion reducing antioxidant power (FRAP) assay, 2,2'-azino-bis(3-ethylbenzothiazoline-6)-sulfonic acid (ABTS) radical scavenging assay, and Fe2+ chelating ability assay were used, respectively, to detect the total polyphenols and 5 kinds of caffeic acid derivatives (chicoric acid, caffeic acid, caftaric acid, chlorogenic acid, and 1,5-dicaffeoylquinic acid) in the roots, stems, leaves, and flowers, and the antioxidant activities of 3 varieties of Echinacea: E. purpurea L., cultivar E. purpurea 'Aloha', and E. purpurea 'White Swan'. RESULTS: E. purpurea L. had the highest contents of total polyphenols, 5 caffeic acid derivatives and antioxidant activities, followed by E. purpurea 'White Swan' and E. purpurea 'Aloha', respectively. E. purpurea 'White Swan' had the strongest ability to remove the DPPH, ABTS•+ and free radicals, and to chelate Fe2+; E. purpurea L. had the strongest ability to reduce FRAP. The correlation analyses revealed that the contents of total polyphenols and caffeic acid derivatives of E. purpurea L. and E. purpurea 'White Swan' were correlated with their antioxidant activities. CONCLUSION: E. purpurea L. was the most appropriate material for the development of medicinal plants. E. purpurea 'White Swan' could be used as a substitute for E. purpurea L. in terms of its antioxidant activity.

Structural elucidation of lactone derivatives from Dendrobium pendulum.

Six lactone derivatives, including four α-pyrones derivatives (1-4), two α-furanone derivatives (5 and 6), were isolated from the Dendrobium pendulum. Structural elucidation of these undescribed lactone derivatives were accomplished on the basis of detailed nuclear magnetic resonance analysis, and the absolute configurations of compounds 1-4 were confirmed by electronic circular dichroism (ECD) techniques. The cytotoxic effects of isolated compounds on human breast cancer cell MDA-MB-231 were evaluated by the MTT assay.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer.

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

Complete 1 H and 13 C assignments of two new sesquiterpenoids from Dendrobium aduncum.

Two new sesquiterpenoids, dendroaduoid A (1) and dendroaduol (2), together with four known sesquiterpenoids were isolated from the stems of Dendrobium aduncum. Their structures were identified by HR-ESI-MS and NMR experiments, and the complete assignments of 1 H and 13 C NMR data for two new sesquiterpenoids were obtained by the aid of HSQC, HMBC, 1 H-1 H COSY, NOESY, and ECD techniques. The cytotoxic effects of the isolated compounds on four tumor cell lines (HCT-116, HepG2, A549, and SW1990) were evaluated using MTT assay. Otherwise, the inhibitory activity of these six sesquiterpenoids on glycosidase was also evaluated.

Development of a Novel 18F-Labeled Probe for PET Imaging of Estrogen Receptor ß.

Estrogen receptor beta (ERß) is an important ER subtype that plays crucial roles in many physiological and pathological disorders. Herein, we developed the probe [18F]PVBO for in vivo ERß targeted PET imaging and obtained promising results. The nonradioactive PVBO showed a 12.5-fold stronger binding affinity to ERß than to ERα in vitro. In vitro assays revealed the specific uptake of [18F]PVBO by DU145 cells. The uptake of [18F]PVBO by DU145 xenografts increased during the 120 min dynamic scanning, with a maximum uptake of 2.80 ± 0.30% ID/g. Based on time activity curves (TACs), the injection of [18F]PVBO with unlabeled PVBO or ERB-041 resulted in a significant signal reduction with the tumor/muscle (T/M) ratio <1 at 30, 60, 75, and 120 min post-injection (p < 0.05). [18F]PVBO demonstrates the feasibility of noninvasively imaging ERß-positive tumors by small-animal PET and provides a new strategy for visualizing ERß in vivo.

Dihydrophenanthrenes and phenanthrenes from Dendrobium terminale.

Two previously undescribed dihydrophenanthrene derivatives (1 and 2) were isolated along with twelve known analogues from the whole plant of Dendrobium terminale. The structures of the new compounds were elucidated on the basis of detailed spectroscopic analysis. The NMR data of known phenanthrene derivatives (7 and 9) were revised by 2D NMR. The isolated compounds were evaluated for cytotoxicity against three kinds of tumor cell lines (sw1990, HCT-116, and HepG2). Especially compounds 11 and 14 showed stronger antitumor effects, and the structure-activity relationship of these compounds was discussed.

Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

Structural and Functional Insights into a Nonheme Iron- and α-Ketoglutarate-Dependent Halogenase That Catalyzes Chlorination of Nucleotide Substrates.

Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical ß-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Diagnostic value of MRI-DWI signal intensity value combined with serum PGI, PGII and CA199 in early gastric cancer.

To explore the diagnostic value of MRI-DWI signal intensity value combined with serum PGI. PGII and CA199 in early gastric cancer. Sixty cases of gastric cancer patients admitted to our hospital from December 2019 to December 2020 were selected as the gastric cancer group and 80 cases of healthy volunteers who underwent physical examination in our hospital during the same period were selected as the healthy group. All the 60 patients underwent MRI-DWI examination, and the pathological diagnosis results were regarded as the gold standard. MRI-DWI images, MRI-DWI signal intensity values of patients with different degrees of gastric cancer differentiation. Serum PGI, PGII and CA199 levels of subjects in the two groups were compared. AUC was used to evaluate the diagnostic value of MRI-DWI signal intensity value combined with serum PGI, PG II and CA199 for early gastric cancer. In the healthy group, T1W1 showed relatively uniform low signal intensity. While T2WI showed no significant increase in signal intensity. In the gastric cancer group. There was diffuse gastric wall thickening, local thickening or mass formation; T1WI and WATS showed slightly lower signal intensity in the lesion area. T2WI, FLAIR and B-TFE showed slightly uneven or moderately increased signal intensity. DWI showed limited diffusion, and the signal intensity increased uniformly or more uniformly, and the range of increase was clear. The signal intensity of MRI-DWI was 89.12 ± 8.14 in patients with low differentiation, 82.17 ± 6.35 in patients with moderate differentiation, and 74.52 ± 4.53 in patients with high differentiation. There were significant differences in the signal intensity of MRI-DWI among the three groups, and the difference was statistically significant (F=12.214, P <0.05). Serum PGI levels of subjects in the gastric cancer group were significantly lower than those in the healthy group, and the levels of PGII and CA199 were significantly higher than that in the healthy group, with statistical significance (P <0.05). The AUC, sensitivity and specificity of MRI-DWI signal intensity value and serum PGI, PGII and CA199 combined indexes in the diagnosis of gastric cancer were significantly higher than those of the independent indexes, with statistical significance (P <0.05). Conclusion: MRI-DWI signal strength value, serum PGI, PGII and CA199 levels are closely related to the occurrence and development of early gastric cancer. The combined detection and diagnosis efficiency is higher, which is helpful to improve the detection rate of early gastric cancer and is worthy of extensive clinical application.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Overview of antiviral drug candidates targeting coronaviral 3C-like main proteases.

Coronaviruses (CoVs) are positive single-stranded RNA viruses that cause severe respiratory syndromes in humans, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Coronavirus disease 2019 (COVID-19) caused by a novel severe acute respiratory syndrome CoV (SARS-CoV-2) at the end of 2019 became a global pandemic. The 3C-like cysteine protease (3CLpro) processes viral polyproteins to yield mature non-structural proteins, thus playing an important role in the CoV life cycle, and therefore is considered as a prominent target for antiviral drugs. To date, many 3CLpro inhibitors have been reported, and their molecular mechanisms have been illustrated. Here, we briefly introduce the structural features of 3CLpro of the human-related SARS-CoV, MERS-CoV and SARS-CoV-2, and explore the potency and mechanism of their cognate inhibitors. This information will shed light on the development and optimization of CoV 3CLpro inhibitors, which may benefit the further designation of therapeutic strategies for treating CoV diseases.

Structure basis of non-structural protein pA151R from African Swine Fever Virus.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded ß-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the ß7-ß8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase.

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.

Biocatalytic synthesis of ginsenoside Rh2 using Arabidopsis thaliana glucosyltransferase-catalyzed coupled reactions.

Ginsenoside Rh2, a rare protopanaxadiol (PPD)-type triterpene saponin isolated from Panax ginseng, exhibits notable anticancer and immune-system-enhancing activities. Glycosylation catalyzed by uridine diphosphate-dependent glucosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rh2. In this study, UGT73C5 isolated from Arabidopsis thaliana was demonstrated to selectively transfer a glucosyl moiety to the C3 hydroxyl group of PPD to synthesize ginsenoside Rh2. UGT73C5 was coupled with sucrose synthase (SuSy) from A. thaliana to regenerate costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The UGT73C5/SuSy ratio, temperature, pH, cofactor UDP, and PPD concentrations for UGT73C5-SuSy coupled reactions were optimized. Through the stepwise addition of PPD, the maximal ginsenoside Rh2 production was 3.2 mg mL-1, which was the highest yield reported to date. These promising results provided an efficient and cost-effective approach to semisynthesize the highly valuable ginsenoside Rh2.

Antagonists for Constitutively Active Mutant Estrogen Receptors: Insights into the Roles of Antiestrogen-Core and Side-Chain.

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both nonpolar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.