Colorectal cancer (CRC) has long been known for its tight association with chronic inflammation, thought to play a key role in tumor onset and malignant progression through the modulation of cancer stemness. However, the underlying molecular and cellular mechanisms are still largely elusive. Here we show that the IL-6/STAT3 inflammatory signaling axis induces the deacetylation of FRA1 at the Lys-116 residue located within its DNA-binding domain. The HDAC6 deacetylase underlies this key modification leading to the increase of FRA1 transcriptional activity, the subsequent transactivation of NANOG expression, and the acquisition of stem-like cellular features. As validated in a large (n = 123) CRC cohort, IL-6 secretion was invariably accompanied by increased FRA1 deacetylation at K116 and an overall increase in its protein levels, coincident with malignant progression and poor prognosis. Of note, combined treatment with the conventional cytotoxic drug 5-FU together with Tubastatin A, a HDAC6-specific inhibitor, resulted in a significant in vivo synergistic inhibitory effect on tumor growth through suppression of CRC stemness. Our results reveal a novel transcriptional and posttranslational regulatory cross-talk between inflammation and stemness signaling pathways that underlie self-renewal and maintenance of CRC stem cells and promote their malignant behavior. Combinatorial treatment aimed at the core regulatory mechanisms downstream of IL-6 may offer a novel promising approach for CRC treatment.

Binding of different ligands to glucocorticoid receptor (GR) may induce different conformational changes and even trigger completely opposite biological functions. To understand the allosteric communication within the GR ligand binding domain, the folding pathway of helix 12 (H12) induced by the binding of the agonist dexamethasone (DEX), antagonist RU486, and modulator AZD9567 are explored by molecular dynamics simulations and Markov state model analysis. The ligands can regulate the volume of the activation function‐2 through the residues Phe737 and Gln738. Without ligand or with agonist binding, H12 swings from inward to outward to visit different folding positions. However, the binding of RU486 or AZD9567 perturbs the structural state, and the passive antagonist state appears more stable. Structure‐based virtual screening and in vitro bioassays are used to discover novel GR ligands that bias the conformation equilibria toward the passive antagonist state. HP‐19 exhibits the best anti‐inflammatory activity (IC50 = 0.041 ± 0.011 µm) in nuclear factor‐kappa B signaling pathway, which is comparable to that of DEX. HP‐19 also does not induce adverse effect‐related transactivation functions of GR. The novel ligands discovered here may serve as promising starting points for the development of GR modulators.

The number of solved G-protein-coupled receptor (GPCR) crystal structures has expanded rapidly, but most GPCR structures remain unsolved. Therefore, computational techniques, such as homology modeling, have been widely used to produce the theoretical structures of various GPCRs for structure-based drug design (SBDD). Due to the low sequence similarity shared by the transmembrane domains of GPCRs, accurate prediction of GPCR structures by homology modeling is quite challenging. In this study, angiotensin II type I receptor (AT1R) was taken as a typical case to assess the reliability of class A GPCR homology models for SBDD. Four homology models of angiotensin II type I receptor (AT1R) at the inactive state were built based on the crystal structures of CXCR4 chemokine receptor, CCR5 chemokine receptor, and δ-opioid receptor, and refined through molecular dynamics (MD) simulations and induced-fit docking, to allow for backbone and side-chain flexibility. Then, the quality of the homology models was assessed relative to the crystal structures in terms of two criteria commonly used in SBDD: prediction accuracy of ligand-binding poses and screening power of docking-based virtual screening. It was found that the crystal structures outperformed the homology models prior to any refinement in both assessments. MD simulations could generally improve the docking results for both the crystal structures and homology models. Moreover, the optimized homology model refined by MD simulations and induced-fit docking even shows a similar performance of the docking assessment to the crystal structures. Our results indicate that it is possible to establish a reliable class A GPCR homology model for SBDD through the refinement by integrating multiple molecular modeling techniques.

Highlights d The lipid A pocket is illustrated in EptA and Ah762 d The MCR-3 inside Linker 59 acts as a facilitator to govern its enzymatic action d The facilitator allows binding of MCR to lipid PE, its first substrate d Linker 59 is a sufficient (rather than prerequisite) element for MCR colistin resistance