Target immobilization on glass microfiber membranes as a label-free strategy for hit identification.

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V2R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V2R were immobilized on the surface of a glass microfiber (GMF) coated with O6-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V2R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V2R ligand treated with SNAP-V2R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the Ki value determined by displacement assays. Furthermore, a Ki prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V2R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V2R@GMF-BG was incubated with the total extract, the ligand that binds to V2R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V2R antagonist. This V2R-immobilized GMF platform can help determine the affinity of V2R-binding hit compounds and screen the compounds efficiently and accurately.

Discovery of novel polysubstituted N-alkyl acridone analogues as histone deacetylase isoform-selective inhibitors for cancer therapy.

Pan-histone deacetylase (HDAC) inhibitors often have some toxic side effects. In this study, three series of novel polysubstituted N-alkyl acridone analogous were designed and synthesised as HDAC isoform-selective inhibitors. Among them, 11b and 11c exhibited selective inhibition of HDAC1, HDAC3, and HDAC10, with IC50 values ranging from 87 nM to 418 nM. However, these compounds showed no inhibitory effect against HDAC6 and HDAC8. Moreover, 11b and 11c displayed potent antiproliferative activity against leukaemia HL-60 cells and colon cancer HCT-116 cells, with IC50 values ranging from 0.56 µM to 4.21 µM. Molecular docking and energy scoring functions further analysed the differences in the binding modes of 11c with HDAC1/6. In vitro anticancer studies revealed that the hit compounds 11b and 11c effectively induced histone H3 acetylation, S-phase cell cycle arrest, and apoptosis in HL-60 cells in a concentration-dependent manner.

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.

Exploring the interactions of antihistamine with retinoic acid receptor beta (RARB) by molecular dynamics simulations and genome-wide meta-analysis.

Kaposi sarcoma (KS) is one of the most common AIDS-related malignant neoplasms, which can leave lesions on the skin among HIV patients. These lesions can be treated with 9-cis-retinoic acid (9-cis-RA), an endogenous ligand of retinoic acid receptors that has been FDA-approved for treatment of KS. However, topical application of 9-cis-RA can induce several unpleasant side effects, like headache, hyperlipidemia, and nausea. Hence, alternative therapeutics with less side effects are desirable. There are case reports associating over-the-counter antihistamine usage with regression of KS. Antihistamines competitively bind to H1 receptor and block the action of histamine, best known for being released in response to allergens. Furthermore, there are already dozens of antihistamines that are FDA-approved with less side effects than 9-cis-RA. This led our team to conduct a series of in-silico assays to determine whether antihistamines can activate retinoic acid receptors. First, we utilized high-throughput virtual screening and molecular dynamics simulations to model high-affinity interactions between antihistamines and retinoic acid receptor beta (RARß). We then performed systems genetics analysis to identify a genetic association between H1 receptor itself and molecular pathways involved in KS. Together, these findings advocate for exploration of antihistamines against KS, starting with our two promising hit compounds, bepotastine and hydroxyzine, for experimental validation study in the future.

Potent Antiplasmodial Derivatives of Dextromethorphan Reveal the Ent-Morphinan Pharmacophore of Tazopsine-Type Alkaloids.

The alkaloid tazopsine 1 was introduced in the late 2000s as a novel antiplasmodial hit compound active against Plasmodium falciparum hepatic stages, with the potential to develop prophylactic drugs based on this novel chemical scaffold. However, the structural determinants of tazopsine 1 bioactivity, together with the exact definition of the pharmacophore, remained elusive, impeding further development. We found that the antitussive drug dextromethorphan (DXM) 3, although lacking the complex pattern of stereospecific functionalization of the natural hit, was harboring significant antiplasmodial activity in vitro despite suboptimal prophylactic activity in a murine model of malaria, precluding its direct repurposing against the disease. The targeted N-alkylation of nor-DXM 15 produced a small library of analogues with greatly improved activity over DXM 3 against P. falciparum asexual stages. Amongst these, N-2'-pyrrolylmethyl-nor-DXM 16i showed a 2- to 36-fold superior inhibitory potency compared to tazopsine 1 and DXM 3 against P. falciparum liver and blood stages, with respectively 760 ± 130 nM and 2.1 ± 0.4 µM IC50 values, as well as liver/blood phase selectivity of 2.8. Furthermore, cpd. 16i showed a 5- to 8-fold increase in activity relative to DXM 3 against P. falciparum stages I-II and V gametocytes, with 18.5 µM and 13.2 µM IC50 values, respectively. Cpd. 16i can thus be considered a promising novel hit compound against malaria in the ent-morphinan series with putative pan cycle activity, paving the way for further therapeutic development (e.g., investigation of its prophylactic activity in vivo).

Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway.

Fangchinoline, a bisbenzylisoquinoline alkaloid extracted from Stephania tetrandra S. Moore, is known to exert anti-cancer activity. A series of new fangchinoline derivatives have been synthesized and evaluated for their anti-cancer activity. In cell viability assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on leukemia and breast cancer cell lines than the parental compound. Among them, 3e exhibited strongest cell growth inhibitory activity in a dose- and time-dependent manner on leukemia cell line HEL through induction of G0/G1 cell cycle arrest and apoptosis. Treatment of HEL cells with 3e also resulted in significant suppression of the MAPK and PI3K/AKT pathway as well as c-MYC downregulation, which may responsible for induction of apoptosis and cell cycle arrest. In docking analysis, high affinity interaction between 3e and Akt1 was responsible for drastic kinase inhibition by the compound. This derivative compound may be useful as a potent anti-cancer agent for treatment of leukemia.