Research and Evaluation of the Allosteric Protein-Specific Force Field Based on a Pre-Training Deep Learning Model.

Allosteric modulators are important regulation elements that bind the allosteric site beyond the active site, leading to the changes in dynamic and/or thermodynamic properties of the protein. Allosteric modulators have been a considerable interest as potential drugs with high selectivity and safety. However, current experimental methods have limitations to identify allosteric sites. Therefore, molecular dynamics simulation based on empirical force field becomes an important complement of experimental methods. Moreover, the precision and efficiency of current force fields need improvement. Deep learning and reweighting methods were used to train allosteric protein-specific precise force field (named APSF). Multiple allosteric proteins were used to evaluate the performance of APSF. The results indicate that APSF can capture different types of allosteric pockets and sample multiple energy-minimum reference conformations of allosteric proteins. At the same time, the efficiency of conformation sampling for APSF is higher than that for ff14SB. These findings confirm that the newly developed force field APSF can be effectively used to identify the allosteric pocket that can be further used to screen potential allosteric drugs based on these pockets.

Identification of Novel CD39 Inhibitors Based on Virtual Screening and Enzymatic Assays.

The accumulation of adenosine in the tumor microenvironment mediates immunosuppression and promotes tumor growth and proliferation. Intervention of the adenosine pathway is an important direction of antitumor immunity research. CD39 is an important ecto-nucleotidases for adenosine generation, therefore targeting the CD39-adenosine pathway is an emerging immune checkpoint for anticancer treatment. However, currently no CD39 inhibitor has been approved by the U.S. Food and Drug Administration. The development of CD39 drugs is urgent for clinical application. In this study, we combined homology modeling, virtual screening, and in vitro enzymatic activity to characterize the structural features of the CD39 protein and identify a triazinoindole-based compound as a CD39 inhibitor. The identified inhibitor and one of its analogues could effectively prevent the enzymatic activity of CD39 with IC50 values of 27.42 ± 5.52 and 79.24 ± 12.21 µM, respectively. At the same time, the inhibitor significantly inhibited the adenosine monophosphate production in colorectal cancer cell lines (HT29 and MC38) and thereafter prevented cell proliferation. Molecular docking studies, mutagenesis, and microscale thermophoresis indicated that residues such as R85 could be the main contributor in binding triazinoindole compounds. The binding mode can potentially be utilized for hit-to-lead optimization, and the identified inhibitor can be further tested for its anticancer activity in vivo or may serve as a chemical agent to study CD39-related functions.

Identification of novel inhibitors of GLUT1 by virtual screening and cell-based assays.

In order to fuel the uncontrolled cell proliferation and division, tumor cells reprogram the energy metabolism to Warburg effect, where glucose is preferably converted by glycolysis even in the presence of oxygen. However, the high energetic demand of tumor cells require upregulating the expression of glucose transporters, notably GLUT1, which substantially increases glucose uptake into cytoplasm. GLUT1 is overexpressed in a variety of tumor cells and is likely to be a potential drug target in the treatment of pan-cancers. Although many small molecules were reported to inhibit the glucose uptake function by various measurements, several shortcomings such as weak binding affinity, low specificity of the known inhibitors demand the identification of alternative inhibitors with novel scaffolds. In this study, we performed a virtual screening campaign by docking each compound from Chemdiv database to the glucose binding pocket based on the crystal structure of GLUT1 (PDB ID 4PYP) and four small molecules with novel scaffolds were identified to inhibit the glucose uptake of cancer cells at the sub-micromole level. The identified compounds may serve as starting points for the development of anti-cancer drugs via the manipulation of the energy metabolism.

Discovery of bilirubin as novel P2X7R antagonist with anti-tumor activity.

As a unique ligand gated ion channel in the P2-receptor family, P2X7R is highly expressed in various tumors. The activated P2X7R facilitates tumor growth and metastasis. Hypoxia, inflammation and necrosis in the tumor microenvironment (TME) cause a large amount of adenosine triphosphate (ATP) accumulated in the TME. High concentration of ATP can abnormally activate P2X7R, which induces pore formation and further facilitates the Ca2+ ion influx and non-specific substance intake. Therefore, inhibition of P2X7R activation can be applied as a potential anti-tumor therapy strategy. However, there is currently no FDA approved drugs for this target for anti-tumor treatment. In this study, we identified bilirubin as novel P2X7R antagonist by using structure based virtual screening combined with cell based assays. Molecular docking studies indicated that bilirubin probably interacted with P2X7R by forming hydrogen-π interactions with residues V173, E174 and K311. The compound bilirubin inhibited the P2X7R gated EB intake by cancer cells. Meanwhile, bilirubin was capable to inhibit the cell proliferation and migration of P2X7R expressed HT29 cells. The phosphorylation of mTOR, STAT3 and GSK3ß were significantly decreased when bilirubin was present. Finally, in vivo experiment exhibited the anti-tumor effect of bilirubin in the MC38 bearing mice model, but did not show tissue damage in different organs. In conclusion, bilirubin was identified as a novel P2X7R antagonist and it may have potential for anti-cancer treatment, although various functions of the molecule should be considered.

Identification of Phelligridin-Based Compounds as Novel Human CD73 Inhibitors.

As an emerging immune checkpoint, CD73 has received more attention in the past decade. Inhibition of CD73 enzymatic activity can enhance antitumor immunity. Several CD73 inhibitors have been identified by in vitro assays in recent years, but they remain premature for clinical application, indicating that more novel CD73 inhibitors should be studied. Herein, we aimed to identify novel CD73 inhibitors that hopefully are suitable drug candidates by using computer-aided drug discovery and enzymatic-based assays. Five-hundred molecules with high binding affinity were retrieved from the Chemdiv-Plus database by using a structure-based virtual screening approach. Then, we analyzed the drug properties of these molecules and obtained 68 small molecules based on the oral noncentral nervous system (CNS) drug profile. The inhibition rates of these molecules against CD73 enzymatic activities were determined at a concentration of 100 µM, and 20 molecules had an inhibition rate greater than 20%, eight of which were dose-dependent, with IC50 values of 6.72-172.1 µM. Among the screening hits, phelligridin-based compounds had the best experimental inhibition values. Modeling studies indicate that the phelligridin group is sandwiched by the rings of F417 and F500 residues. The identified inhibitors have a molecular weight of approximately 500 Dal and are predicted to form primarily hydrogen bonds with CD73 in addition to hydrophobic stacking interactions. In conclusion, novel inhibitors with satisfactory drug properties may serve as lead compounds for the development of CD73-targeting drugs, and the binding modes may provide insight for phelligridin-based drug design.

Identification of a novel GLUT1 inhibitor with in vitro and in vivo anti-tumor activity.

Glucose transporter (GLUT) is a group of membrane proteins which transport extracellular glucoses into cytoplasm, amongst GLUT1 is widely up-regulated in tumor cells. However, no FDA approved GLUT drug has been developed. In this study, we synthesized and identified a novel GLUT1 inhibitor (SMI277) based on in vitro assays and in vivo experiments. Compared with a known GLUT1 inhibitor, SMI277 showed stronger inhibitory activity to glucose uptake, and the inhibition was increased by 40 %. Lactate secretions were decreased by SMI277 in a dose dependent manner. SMI277 was able to inhibit cell proliferations and induce apoptosis of tumor cells. Compared to that of the control group, the tumor growth in mouse model with the administration of 10 mg/kg SMI277 was significantly alleviated and the tumor size was reduced by 58 % on day 21 after inoculation. Interestingly, SMI277 could negatively regulate the expression of GLUT1 protein. Ex vivo experiments showed that SMI277 was capable to enhance CD8+ T cell response. Residues Q283, F379 and E380 were identified as contact residues for GLUT1/SMI277 interactions by mutagenesis based binding affinity measurement. In conclusion, SMI277 appeared to be a good lead compound for drug development with specific GLUT1+ cancer treatment.

Identification of novel P2X7R antagonists by using structure-based virtual screening and cell-based assays.

In the tumor microenvironment, inflammation and necrosis cause the accumulations of ATP extracellularly, and high concentrations of ATP can activate P2X7 receptors (P2X7R), which leads to the influx of Na+ , K+ , or Ca2+ into cells and trigger the downstream signaling pathways. P2X7R is a relatively unique ligand-gated ion channel, which is over-expressed in most tumor cells. The activated P2X7R facilitates the tumor growth, invasion, and metastasis. Inhibition of the P2X7R activation can be applied as a potential anti-tumor therapy strategy. There are currently no anti-tumor agents against P2X7R, though several P2X7R antagonists for indications such as anti-inflammatory and anti-depression were reported. In this study, we combined homology modeling (HM), virtual screening, and EB intake assay to characterize the structural features of P2X7R and identify several novel antagonists, which were chemically different from any other known P2X7R antagonists. The identified antagonists could effectively prevent the pore opening of P2X7R with IC50 values ranging from 29.14 to 35.34 µM. HM model showed the area between ATP-binding pocket, and allosteric sides were hydrophobic and suitable for small molecule interaction. Molecular docking indicated a universal binding mode, of which residues R294 and K311 were used as hydrogen bond donors to participate in antagonist interactions. The binding mode can potentially be utilized for inhibitor optimization for increased affinity, and the identified antagonists can be further tested for anti-cancer activity or may serve as chemical agents to study P2X7R related functions.