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.

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.

ESR1 F404 Mutations and Acquired Resistance to Fulvestrant in ESR1-Mutant Breast Cancer.

Fulvestrant is used to treat patients with hormone receptor-positive advanced breast cancer, but acquired resistance is poorly understood. PlasmaMATCH Cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Baseline ESR1 mutations Y537S are associated with poor outcomes and Y537C with good outcomes. Sequencing of baseline and EOT ctDNA samples (n = 69) revealed 3/69 (4%) patients acquired novel ESR1 F404 mutations (F404L, F404I, and F404V), in cis with activating mutations. In silico modeling revealed that ESR1 F404 contributes to fulvestrant binding to estrogen receptor-alpha (ERα) through a pi-stacking bond, with mutations disrupting this bond. In vitro analysis demonstrated that single F404L, E380Q, and D538G models were less sensitive to fulvestrant, whereas compound mutations D538G + F404L and E380Q + F404L were resistant. Several oral ERα degraders were active against compound mutant models. We have identified a resistance mechanism specific to fulvestrant that can be targeted by treatments in clinical development. SIGNIFICANCE: Novel F404 ESR1 mutations may be acquired to cause overt resistance to fulvestrant when combined with preexisting activating ESR1 mutations. Novel combinations of mutations in the ER ligand binding domain may cause drug-specific resistance, emphasizing the potential of similar drug-specific mutations to impact the efficacy of oral ER degraders in development. This article is featured in Selected Articles from This Issue, p. 201.

Characterization of bacteria swarming effect under plasmonic optical fiber illumination.

Significance: Plasmo-thermo-electrophoresis (PTEP) involves using plasmonic microstructures to generate both a large-scale convection current and a near-field attraction force (thermo-electrophoresis). These effects facilitate the collective locomotion (i.e., swarming) of microscale particles in suspension, which can be utilized for numerous applications, such as particle/cell manipulation and targeted drug delivery. However, to date, PTEP for ensemble manipulation has not been well characterized, meaning its potential is yet to be realized. Aim: Our study aims to provide a characterization of PTEP on the motion and swarming effect of various particles and bacterial cells to allow rational design for bacteria-based microrobots and drug delivery applications. Approach: Plasmonic optical fibers (POFs) were fabricated using two-photon polymerization. The particle motion and swarming behavior near the tips of optical fibers were characterized by image-based particle tracking and analyzing the spatiotemporal concentration variation. These results were further correlated with the shape and surface charge of the particles defined by the zeta potential. Results: The PTEP demonstrated a drag force ranging from a few hundred fN to a few tens of pN using the POFs. Furthermore, bacteria with the greater (negative) zeta potential (|ζ|>10 mV) and smoother shape (e.g., Klebsiella pneumoniae and Escherichia coli) exhibited the greatest swarming behavior. Conclusions: The characterization of PTEP-based bacteria swarming behavior investigated in our study can help predict the expected swarming behavior of given particles/bacterial cells. As such, this may aid in realizing the potential of PTEP in the wide-ranging applications highlighted above.

Effect of Manufacture-Induced Interfaces on the Tensile Properties of 3D Printed Polyamide and Short Carbon Fibre-Reinforced Polyamide Composites.

This study aims to elucidate the structure-property-process relationship of 3D printed polyamide and short carbon fibre-reinforced polyamide composites. The macroscopic properties (tensile modulus) of the 3D printed samples are quantitatively correlated to the printing process-induced intrinsic microstructure with multiple interfaces. The samples were printed with different layer thicknesses (0.1, 0.125 and 0.2 mm) to obtain the varied number of interface densities (number of interfaces per unit sample thickness). The result shows that the printed short carbon fibre-reinforced polyamide composites had inferior partially bonded interfaces compared to the printed polyamide, and consequently exhibited interface-dependent elastic performance. The tensile modulus of 3 mm thick composites decreased up to 18% as a function of interface density, whilst the other influencing aspects including porosity, crystallinity and fibre volume fraction (9%) were the same. Injection moulding was also employed to fabricate samples without induced interfaces, and their tensile properties were used as a benchmark. Predictions based on the shear-lag model were in close agreement (<5%) with the experimental data for the injection-moulded composites, whereas the tensile modulus of the printed composites was up to 38% lower than the predicted modulus due to the partial bonded interfaces.

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.

Cosmic-ray neutron fluxes and spectra at different altitudes based on Monte Carlo simulations.

Cosmic-ray neutrons (CRNs) account for about half of the radiation dose received by airline crews and passengers at aviation altitudes. CRNs also comprise important background radiation near the ground, which should be considered in neutron counts for the purpose of nuclear safeguarding and homeland security. In this study, simulations were conducted with the Geant4 toolkit to describe the air shower of cosmic rays. The latest experimental driving models for the atmosphere, geomagnetic field, and primary galactic cosmic rays were applied in these simulations. The simulation was validated by comparing the results with those measured on the NASA ER-2 aircraft. the CRN fluxes and spectra were calculated at altitudes ranging from one to tens of kilometers. We determined the characteristics of the CRN spectra and analyzed their dependency on the altitude. To consider their impact on the local environment, the CRNs near the ground were modeled with a specific equivalent approach, which allowed the simulations to be conducted with limited computer processing power. The parameters for the incident primary CRNs near the ground were calculated by simulating the cosmic ray air shower. The modeling dimensions were considered for the air and ground, and an appropriate approximation solution was obtained. The model near the ground was used to investigate the dependences of the CRN flux and spectrum on the soil moisture.

Development of novel phenoxy-diketopiperazine-type plinabulin derivatives as potent antimicrotubule agents based on the co-crystal structure.

The co-crystal structure of Compound 6b with tubulin was prepared and solved for indicating the binding mode and for further optimization. Based on the co-crystal structures of tubulin with plinabulin and Compound 6b, a total of 27 novel A/B/C-rings plinabulin derivatives were designed and synthesized. Their biological activities were evaluated against human lung cancer NCI-H460 cell line. The optimum phenoxy-diketopiperazine-type Compound 6o exhibited high potent cytotoxicity (IC50 = 4.0 nM) through SAR study of three series of derivatives, which was more potent than plinabulin (IC50 = 26.2 nM) and similar to Compound 6b (IC50 = 3.8 nM) against human lung cancer NCI-H460 cell line. Subsequently, the Compound 6o was evaluated against other four human cancer cell lines. Both tubulin polymerization assay and immunofluorescence assay showed that Compound 6o could inhibit microtubule polymerization efficiently. Furthermore, theoretical calculation of the physical properties and molecular docking were elucidated for these plinabulin derivatives. The binding mode of Compound 6o was similar to Compound 6b based on the result of molecular docking. The theoretical calculated LogPo/w and PCaco of Compound 6o were better than Compound 6b, which could enhance its cytostatic activity. Therefore, Compound 6o might be developed as a novel potent anti-microtubule agent.

In vitro and in vivo pharmacokinetic and pharmacodynamic study of MBRI-001, a deuterium-substituted plinabulin derivative as a potent anti-cancer agent.

MBRI-001 was demonstrated preliminary better pharmacokinetics and antitumor effects than that of plinabulin in vivo. In this approach, we further carried out systematic pharmacokinetic and pharmacodynamic study of MBRI-001 in vitro and in vivo. MBRI-001 was tested stable in rat plasma and more stable in liver microsomes than plinabulin in vitro. In vivo, MBRI-001 could be distributed rapidly and widely in various tissues, especially the concentration of MBRI-001 in lung was remarkably higher than other tissues. Excretion study indicated that MBRI-001 might been decomposed and excreted as metabolites. Additionally, the combination treatment of MBRI-001 and gefitinib revealed better antitumor inhibition rate than monotherapy in vivo. Therefore, we suggest that MBRI-001 could be developed as a promising anti-cancer agent in near future.

Impact of long-term potassium supplementation on thiazide diuretic-induced abnormalities of glucose and uric acid metabolisms.

Treatment of hypertension with thiazide diuretics may trigger hypokalemia, hyperglycemia, and hyperuricemia. Some studies suggest simultaneous potassium supplementation in hypertensive patients using thiazide diuretics. However, few clinical studies have reported the impact of long-term potassium supplementation on thiazide diuretic-induced abnormalities in blood glucose and uric acid (UA) metabolisms. One hundred hypertensive patients meeting the inclusion criteria were equally randomized to two groups: IND group receiving indapamide (1.25-2.5 mg daily) alone, and IND/KCI group receiving IND (1.25-2.5 mg daily) plus potassium chloride (40 mmol daily), both for 24 weeks. At the end of 24-week follow-up, serum K+ level in IND group decreased from 4.27 ± 0.28 to 3.98 ± 0.46 mmol/L (P < 0.001), and fasting plasma glucose (FPG) and UA increased from 5.11 ± 0.52 to 5.31 ± 0.57 mmol/L (P < 0.05), and from 0.404 ± 0.078 to 0.433 ± 0.072 mmol/L (P < 0.05), respectively. Serum K+ level in IND/KCl group decreased from 4.27 ± 0.36 to 3.89 ± 0.28 mmol/L (P < 0.001), and FPB and UA increased from 5.10 ± 0.41 to 5.35 ± 0.55 mmol/L (P < 0.01), and from 0.391 ± 0.073 to 0.457 ± 0.128 mmol/L (P < 0.001), respectively. The difference value between the serum K+ level and FPG before and after treatment was not statistically significant between the two groups. However, the difference value in UA in IND/KCl group was significantly higher than that in IND group (0.066 (95% confidence interval (CI): 0.041-0.090) mmol/L vs. 0.029 (95% CI: 0.006-0.058) mmol/L, P < 0.05). The results showed that long-term routine potassium supplementation could not prevent or attenuate thiazide diuretic-induced abnormalities of glucose metabolism in hypertensive patients; rather, it may aggravate the UA metabolic abnormality.

Design, synthesis and biological evaluation of anti-pancreatic cancer activity of plinabulin derivatives based on the co-crystal structure.

Based on the co-crystal structures of tubulin with plinabulin and Compound 1 (a derivative of plinabulin), a total of 18 novel plinabulin derivatives were designed and synthesized. Their biological activities were evaluated against human pancreatic cancer BxPC-3 cell lines. Two novel Compounds 13d and 13e exhibited potent activities with IC50 at 1.56 and 1.72 nM, respectively. The tubulin polymerization assay indicated that these derivatives could inhibit microtubule polymerization. Furthermore, the interaction between tubulin and these compounds were elucidated by molecular docking. The binding modes of Compounds 13d and 13e were similar to the co-crystal structure of Compound 1. H-π interaction was observed between the aromatic hydrogen of thiophene moiety with Phe20, which could enhance their binding affinities.

Synthesis of deuterium-enriched and fluorine-substituted plinabulin derivatives and evaluation of their antitumor activities.

Deuterium-enriched and fluorine-substituted compounds have been widely applied in drug discovery due to their advantages in the studies of clinical pharmacokinetics and metabolic profiles. Herein we synthesized a library of deuterated and fluorine-substituted plinabulin derivatives, and all 15 D- or F-compounds were characterized by MS, [Formula: see text] NMR and [Formula: see text]. Their antitumor activities were evaluated against human Jurkat T lymphocyte cells.

Development of MBRI-001, a deuterium-substituted plinabulin derivative as a potent anti-cancer agent.

Plinabulin, a drug targeting microtubule of cancer cells, has been currently tried in its phase III clinical study. However, low efficacy caused by poor pharmacokinetic (PK) properties has been considered to be the main obstacle to approved by the Food and Drug Administration. Herein, we introduced a deuterium atom as an isostere in its structure to become a new compound named (MBRI-001, No. 9 in a series of deuterium-substituted compounds). The structure of MBRI-001 was characterized by HRMS, NMR, IR and a single crystal analysis. MBRI-001 exhibited better pharmacokinetic characteristics than that of plinabulin. Additionally, its antitumor activity is in a low nanomolar level for a variety of cancer cell lines and high activity against human NCI-H460 xenograted in mice intravenous administration. Importantly, continuous administration of MBRI-001 exhibited lower toxicity compared to docetaxel. We thus suggest that MBRI-001 could be developed as a promising anti-cancer agent in near future.

Iodine(III)-mediated [3 + 2] cyclization for one-pot synthesis of benzo[d]isoxazole-4,7-diols in aqueous medium.

A one-pot [3 + 2] cycloadditive synthesis of benzo[d]isoxazole-4,7-diols in aqueous medium was carried out via nitrile oxides and benzoquinone intermediates by taking advantage of iodobenzene diacetate as an oxidant. This method can also be used to synthesize benzodiisoxazole-4,8-diols, isoxazolo[5,4-a]phenazines, and indazole-4,7-diols, which are difficult to obtain by classical methods.

First synthesis of 12-oxosoladulcidine and its derivatives as potential antitumor steroidal alkaloids.

The first synthesis of 12-oxosoladulcidine (= (3beta,5alpha,22alpha,25R)-3-hydroxyspirosolan-12-one; 4) is reported, and its structure was confirmed by single-crystal X-ray diffraction analysis. Compound 4 was readily obtained in five steps in an overall yield of 31%, starting from hecogenin (5). By slightly modifying the synthetic protocol, eight analogues of 4 were also prepared. The title compound and its derivatives are expected to be potent antitumor alkaloids, since structurally closely related to the known antitumor agents soladulcidine (2) and hecogenin (5).