Triple-negative breast cancer (TNBC) is the most aggressive and lethal subtype of breast cancer among women. Chemotherapy acts as the standard regimen for TNBC treatment but suffers from limited drug accumulation in tumor regions and undesired side effects. Herein, we developed a synergistic strategy by combining a red blood cell (RBC) membrane-liposome hybrid nanovesicle with short-term fasting (STF) for improved chemotherapy of TNBC. The biomimetic nanovesicles exhibited reduced phagocytosis by macrophages while displaying a significant increase in tumor cell uptake through caveolae/raft-mediated endocytosis under nutrient-deprivation conditions. Importantly, drug-loaded nanovesicles and STF treatment synergistically increased the cytotoxicity of tumor cells by inhibiting their cell cycles and aerobic glycolysis as well as amplifying the reactive oxygen species (ROS) and autophagosomes generation. In the STF-treated mice, biomimetic nanovesicles greatly improved the antitumor efficacy at a lower drug dosage and inhibited the undesired metastasis of TNBC. Overall, we demonstrated that biomimetic nanovesicles synergizing with STF therapy serve as a promising therapeutic strategy for enhanced chemotherapy of malignant TNBC.

Vaccines provide a powerful tool to modulate the immune system for human disease prevention and treatment. Classical vaccines mainly initiate immune responses in the lymph nodes (LNs) after subcutaneous injection. However, some vaccines suffer from inefficient delivery of antigens to LNs, undesired inflammation, and slow immune induction when encountering the rapid proliferation of tumors. Alternatively, the spleen, as the largest secondary lymphoid organ with a high density of antigen-presenting cells (APCs) and lymphocytes, acts as an emerging target organ for vaccinations in the body. Upon intravenous administration, the rationally designed spleen-targeting nanovaccines can be internalized by the APCs in the spleen to induce selective antigen presentation to T and B cells in their specific sub-regions, thereby rapidly boosting durable cellular and humoral immunity. Herein, the recent advances of spleen-targeting nanovaccines for immunotherapy based on the anatomical architectures and functional zones of the spleen, as well as their limitations and perspectives for clinical applications are systematically summarized. The aim is to emphasize the design of innovative nanovaccines for enhanced immunotherapy of intractable diseases in the future.

Three structurally closely related dopamine D1 receptor positive allosteric modulators (D1 PAMs) based on a tetrahydroisoquinoline (THIQ) scaffold were profiled for their CYP3A4 induction potentials. It was found that the length of the linker at the C5 position greatly affected the potentials of these D1 PAMs as CYP3A4 inducers, and the level of induction correlated well with the activation of the pregnane X receptor (PXR). Based on the published PXR X-ray crystal structures, we built a binding model specifically for these THIQ-scaffold-based D1 PAMs in the PXR ligand-binding pocket via an ensemble docking approach and found the model could explain the observed CYP induction disparity. Combined with our previously reported D1 receptor homology model, which identified the C5 position as pointing toward the solvent-exposed space, our PXR-binding model coincidentally suggested that structural modifications at the C5 position could productively modulate the CYP induction potential while maintaining the D1 PAM potency of these THIQ-based PAMs.

Background: Lung adenocarcinoma (LUAD) shows intratumoral heterogeneity, a highly complex phenomenon that known to be a challenge during cancer therapy. Considering the key role of monocytic myeloid-derived suppressor cells (M-MDSCs) in the tumor microenvironment (TME), we aimed to build a prognostic risk model using M-MDSCs-related genes.Methods: M-MDSCs-related genes were extracted from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Utilized univariate survival analysis and random forest algorithm to screen candidate genes. A least absolute shrinkage and selection operator (LASSO) Cox regression analysis was selected to build the risk model. Patients were scored and classified into high- and low-risk groups based on the median risk scores. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis along with R packages "estimate " and "ssGSEA " were performed to reveal the mechanism of risk difference. Prognostic biomarkers and tumor mutation burden (TMB) were combined to predict the prognosis. Nomogram was carried out to predict the survival probability of patients in 1, 3, and 5 years.Results: 8 genes (VPREB3, TPBG, LRFN4, CD83, GIMAP6, PRMT8, WASF1, and F12) were identified as prognostic biomarkers. The GEO validation dataset demonstrated the risk model had good generalization effect. Significantly enrichment level of cell cycle-related pathway and lower content of CD8(+) T cells infiltration in the high-risk group when compared to low-risk group. Morever, the patients were from the intersection of high-TMB and low-risk groups showed the best prognosis. The nomogram demonstrated good consistency with practical outcomes in predicting the survival rate over 1, 3, and 5 years.Conclusion: The risk model demonstrate good prognostic predictive ability. The patients from the intersection of low-risk and high-TMB groups are not only more sensitive response to but also more likely to benefit from immune-checkpoint-inhibitors (ICIs) treatment.

Results from recently completed clinical studies suggest the dopamine D1 receptor positive allosteric modulator (PAM) mevidalen (1) could offer unique value for lewy body dementia (LBD) patients. In nonclinical assessments, 1 was mainly eliminated by CYP3A4-mediated metabolism, therefore at the risk of being a victim of drug-drug interactions (DDI) with CYP3A4 inhibitors and inducers. An effort was initiated to identify a new D1 PAM with an improved DDI risk profile. While attempts to introduce additional metabolic pathways mediated by other CYP isoforms failed to provide molecules with an acceptable profile, we discovered that the relative contribution of CYP-mediated oxidation and UGT-mediated conjugation could be tuned to reduce the CYP3A4-mediated victim DDI risk. We have identified LY3154885 (5), a D1 PAM that possesses similar in vitro and in vivo pharmacologic properties as 1, but is metabolized mainly by UGT, predicting it could potentially offer lower victim DDI risk in clinic.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Insulin receptor (IR) signaling defects cause a variety of metabolic diseases including diabetes. Moreover, inherited mutations of the IR cause severe insulin resistance, leading to early morbidity and mortality with limited therapeutic options. A previously reported selective IR agonist without sequence homology to insulin, S597, activates IR and mimics insulin’s action on glycemic control. To elucidate the mechanism of IR activation by S597, we determine cryo-EM structures of the mouse IR/S597 complex. Unlike the compact T-shaped active IR resulting from the binding of four insulins to two distinct sites, two S597 molecules induce and stabilize an extended T-shaped IR through the simultaneous binding to both the L1 domain of one protomer and the FnIII-1 domain of another. Importantly, S597 fully activates IR mutants that disrupt insulin binding or destabilize the insulin-induced compact T-shape, thus eliciting insulin-like signaling. S597 also selectively activates IR signaling among different tissues and triggers IR endocytosis in the liver. Overall, our structural and functional studies guide future efforts to develop insulin mimetics targeting insulin resistance caused by defects in insulin binding and stabilization of insulin-activated state of IR, demonstrating the potential of structure-based drug design for insulin-resistant diseases.

The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2 to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over β-arrestin recruitment. Ex vivo experiments show that the combination of GLP-1(9-36)NH2 and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.

Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a 'molecular glue'. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric G(s). The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein-protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

Analogs based on the 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate scaffold showed high potency and selectivity as both group II mGlu receptors orthosteric agonists and antagonists. This scaffold was initially designed to mimic the fully extended glutamate backbone conformation that was hypothesized to be the active conformation for the group II mGlu receptors. With the availability of crystal structures of l-Glu-bound amino terminal domain proteins from multiple mGlu receptor subtypes spanning all three subgroups, a new steric hindrance hypothesis was proposed to account for the scaffold's unique group II selectivity that explores the subtle distance differences between the α-carbon of l-Glu and the center of the tyrosine phenyl ring from the bottom lobe (e.g. Y216 of mGlu2).

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

The amino terminal domain (ATD) of the metabotropic glutamate (mGlu) receptors contains the orthosteric glutamate recognition site, which is highly conserved across the eight mGlu receptor subtypes. In total, 29 X-ray crystal structures of the mGlu ATD proteins have been reported to date. These structures span across 3 subgroups and 6 subtypes, and include apo, agonist- and antagonist-bound structures. We will discuss the insights gained from the analysis of these structures with the focus on the interactions contributing to the observed group and subtype selectivity for select agonists. Furthermore, we will define the full expanded orthosteric ligand binding pocket (LBP) of the mGlu receptors, and discuss the macroscopic features of the mGlu ATD proteins.

( S)-3,4-Dicarboxyphenylglycine (DCPG) was first reported in 2001 as a potent orthosteric agonist with high subtype selectivity for the mGlu8 receptor, but the structural basis for its high selectivity is not well understood. We have solved a cocrystal structure of recombinant human mGlu8 amino terminal domain (ATD) protein bound to ( S)-DCPG, which possesses the largest lobe opening angle observed to date among known agonist-bound mGlu ATD crystal structures. The binding conformation of ( S)-DCPG observed in the crystal structure is significantly different from that in the homology model built from an l-glutamate-bound rat mGlu1 ATD crystal structure, which has a smaller lobe opening angle. This highlights the importance of considering various lobe opening angles when modeling mGlu ATD-ligand complex. New homology models of other mGlu receptors based on the ( S)-DCPG-bound mGlu8 ATD crystal structure were explored to rationalize ( S)-DCPG's high mGlu8 receptor subtype selectivity.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4β-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4 contribute to its exquisite Group III functional agonist potency and selectivity.

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.