| 1. |
- Battisti, Umberto Maria, et al.
(författare)
-
Ellagic Acid and Its Metabolites as Potent and Selective Allosteric Inhibitors of Liver Pyruvate Kinase
- 2023
-
Ingår i: Nutrients. - : MDPI AG. - 2072-6643. ; 15:3
-
Tidskriftsartikel (refereegranskat)abstract
- Liver pyruvate kinase (PKL) has recently emerged as a new target for non-alcoholic fatty liver disease (NAFLD), and inhibitors of this enzyme could represent a new therapeutic option. However, this breakthrough is complicated by selectivity issues since pyruvate kinase exists in four different isoforms. In this work, we report that ellagic acid (EA) and its derivatives, present in numerous fruits and vegetables, can inhibit PKL potently and selectively. Several polyphenolic analogues of EA were synthesized and tested to identify the chemical features responsible for the desired activity. Molecular modelling studies suggested that this inhibition is related to the stabilization of the PKL inactive state. This unique inhibition mechanism could potentially herald the development of new therapeutics for NAFLD.
|
|
| 2. |
- Battisti, U. M., et al.
(författare)
-
Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase
- 2023
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 24:1
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50=0.29 μM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.
|
|
| 3. |
- Gao, Chunxia, et al.
(författare)
-
Analysis of Biphenyl-Type Inhibitors Targeting the Eg5 alpha 4/alpha 6 Allosteric Pocket
- 2017
-
Ingår i: Acs Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:5, s. 1836-1849
-
Tidskriftsartikel (refereegranskat)abstract
- Eg5 is a mitotic kinesin protein that plays an important role in the formation and maintenance of the bipolar spindle during the mitotic phase. Due to its potentially reduced side effects in cancer therapy, Eg5 is considered to be an attractive target for developing anticancer inhibitors. Herein, we report a computational modeling study involving biphenyl-type inhibitors known to interact with the a4/a6 allosteric pocket of Eg5. Compared to the well-known a2/L5/a3 allosteric inhibitors, biphenyltype inhibitors show a unique activity profile. In the Eg5-PVZB1194 (a biphenyl-type inhibitor) crystal structure, loop L11, which is located in the entrance of the alpha 4/alpha 6 allosteric-binding pocket, is missing due to crystal-packing effects. To better understand the role of this flexible loop upon biphenyl-type inhibitor-binding, MD simulations were performed to observe the L11 conformations from different states. It was demonstrated that L11 was more stabilized and showed less fluctuation when PVZB1194 was bound to Eg5. Residue Asn287 from L11 forms hydrogen bonding to the sulfone group of PVZB1194, whereby L11 moves inward to the alpha 4/alpha 6 allosteric pocket and moves away from the pocket in absence of the inhibitor. Pharmacophore, three-dimensional (3D)-QSAR, and ADME studies of biphenyl-type inhibitors of Eg5 were also performed. A best pharmacophore model, DDRRH. 6, was generated, having correlation coefficients in the 3D-QSAR study of R-2 = 0.81 and Q(2) = 0.64. Furthermore, docking studies were carried out to observe the interaction between the remaining biphenyl-type inhibitors with Eg5. In addition, on the basis of fragment docking, a structure-based pharmacophore was generated, which shares good overlap of the DHRR features of the pharmacophore model DDHRR. 6. The structure-based pharmacophore also contains extra hydrogen-bond acceptors and hydrophobic groups, features which provide possibilities in developing new or improved series of compounds.
|
|
| 4. |
- Gao, Chunxia, et al.
(författare)
-
Characterization of interactions and pharmacophore development for DFG-out inhibitors to RET tyrosine kinase
- 2015
-
Ingår i: Journal of Molecular Modeling. - : Springer Science and Business Media LLC. - 1610-2940 .- 0948-5023. ; 21:7
-
Tidskriftsartikel (refereegranskat)abstract
- RET (rearranged during transfection) tyrosine kinase is a promising target for several human cancers. Abt-348, Birb-796, Motesanib and Sorafenib are DFG-out multi-kinase inhibitors that have been reported to inhibit RET activity with good IC50 values. Although the DFG-out conformation has attracted great interest in the design of type II inhibitors, the structural requirements for binding to the RET DFG-out conformation remains unclear. Herein, the DFG-out conformation of RET was determined by homology modelling, the four inhibitors were docked, and the binding modes investigated by molecular dynamics simulation. Binding free energies were calculated using the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method. The trends in predicted binding free affinities correlated well with experimental data and were used to explain the activity difference of the studied inhibitors. Per-residue energy decomposition analyses provided further information on specific interaction properties. Finally, we also conducted a detailed e-pharmacophore modelling of the different RET-inhibitor complexes, explaining the common and specific pharmacophore features of the different complexes. The results reported herein will be useful in future rational design of novel DFG-out RET inhibitors.
|
|
| 5. |
- Gao, Chunxia
(författare)
-
Computer-aided drug design approaches in developing anti-cancer inhibitors
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thesis entitled “computer-aided drug design approaches in developing anti-cancer drug” is divided into a total of six chapters. In the first chapter, an overview of drug discovery and development are introduced. Nowadays, drug discovery and development has clearly changed the course of the disease treatment, numerous of therapeutic agents have been designed to treat cancers, cardiovascular diseases, infections etc. However, the whole process of drug discovery and development is lengthy and complicated, which require huge input of time, money, and resources. The whole process can be divided into five main stages, including target identification, lead discovery and optimization, preclinical tests and clinical trials. Acknowledging that drug discovery is complicated in general, oncology has one of the poorest records for developing into clinical trial phases, however, we have moved into a new “golden era” for cancer drug development through molecular targeting recently. In the second chapter, computer-aided drug design (CADD), which is effective methods for facilitating and expediting drug discovery and development process, are introduced. Some approved drugs that credited their discovery in large part to the tools of CADD were reported, and the application of CADD has extended to two directions in drug discovery and development: upstream for target identification and validation, and downstream for preclinical study, mostly ADME/T prediction. Though CADD holds a great promise for future progress in drug discovery and development, it is still an evolving technology and has number of limitations that should be resolved. In the third chapter, a detailed introduction of the anti-cancer targets in this thesis is given. The targets include tyrosine kinase RET, kinesin Eg5 and KIF18B, histone transfeRase Tip60 and the GTPase K-Ras. The involving signalling pathway, the cancer induced mechanism and the current available inhibitors for the targets are discussed. In the fourth chapter, the methodologies applied for different projects will be discussed, including homology modelling, docking, molecular dynamics simulations, structure-based pharmacophore, ligand-based pharmacophore and 3D-QSAR, and density functional theory. The detailed theorectical background and the main steps involve in the use of these methods are outlined. In the fifth chapter, I provide a summary of seven papers or manuscripts, which are related to this thesis. Based on the RET target, we have studied the interaction and pharmacophore of four DFG-out inhibitors, mutations in the cadherin like domain, and prediction and calculation of a photo-switchable inhibitor. An inhibitor was designed and validated for Tip60. Biphenyl-type inhibitors targeting Eg5 were fully investigated from different perspectives. Inhibitors were designed to target the α4/α6 allosteric pocket of KIF18B, in order to investigate the specific molecular functional roles of KIF18. Mutations on the binding interface of K-Ras and GAP were looked into and the continuous active oncogenic signalling caused by specific mutations was explained. In the final chapter, conclusions and future perspective is given, CADD is indeed a very useful tool for pharmaceutical companies and academic research groups to search for potential drug candidates, meanwhile, it is also necessary to improve the current CADD methods. The different projects included in this thesis have the potential for further development.
|
|
| 6. |
- Gao, Chunxia, et al.
(författare)
-
Defects in the calcium-binding region drastically affect the cadherin-like domains of RET tyrosine kinase
- 2016
-
Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 18:12, s. 8673-8681
-
Tidskriftsartikel (refereegranskat)abstract
- Mutations in the rearranged during transfection (RET) tyrosine kinase gene leading to gain or loss of function have been associated with the development of several human cancers and Hirschsprung's disease (HSCR). However, to what extent these mutations affect individual bio-molecular functions remains unclear. In this article, the functionally significant mutations in the RET CLD1-4 calcium-binding site which lead to HSCR, and depletion of calcium ions in the RET CLD1-4 calcium binding site, were investigated by molecular dynamics simulations - to understand the mechanistic action of the mutations or loss of calcium ions in altering the protein kinase structure, dynamics, and stability. The mutations or loss of calcium ions change the local conformation and change the free energy landscape. Specifically, the mutations and loss of calcium ions decrease the radius of gyration of the whole structure, leading to improper protein folding and GFL-GFR alpha contact site reduction. Furthermore, based on the most populated conformation in the wildtype MD simulations, a pharmacophore was generated by fragment docking to identify key features of the possible inhibitors targeting the calcium binding site. Overall, the findings may provide useful structural insights into the molecular mechanism underlying RET calcium-binding site mutations and assist in development of novel drugs targeting the extracellular ligand contact site of wildtype RET.
|
|
| 7. |
- Gao, Chunxia, et al.
(författare)
-
Impact of mutations on K-Ras-p120GAP interaction
- 2013
-
Ingår i: Computational Molecular Biosciences. - : Scientific Research Publishing, Inc.. - 2165-3445 .- 2165-3453. ; 3:2, s. 9-17
-
Tidskriftsartikel (refereegranskat)abstract
- The K-Ras protein plays a key role in the signal transduction cascade. Certain mutations in K-Ras lead to a permanent “on” state which results in tumorigenesis due to failed interaction with the GTPase activating protein (GAP). In this study, we examined the mutations E31N, D33N and D38N of K-Ras coupled and decoupled to wildtype GAP-334 and mutation K935N of GAP-334 coupled and decoupled to wildtype K-Ras, to illustrate the potential mechanism by which these mutants affect the interaction between the two proteins. We identify Tyr32 in the Ras Switch I region as a critical residue that acts as a gate to the GTP binding site and which needs to be “open” during Ras coupling with GAP to allow for insertion of GAP residue Arg789. This residue plays a vital role in stabilizing the transition state during GTP hydrolysis. The different mutations studied herein caused a reduced binding affinity, and the fluctuation of the Tyr32 side chain might hinder the insertion of Arg789. This may in turn be the cause of decreased GTP hydrolysis, and permanent “on” state of K-Ras, observed for these mutants.
|
|
| 8. |
- Gao, Chunxia, et al.
(författare)
-
Rational design and validation of a Tip60 histone acetyltransferase inhibitor
- 2014
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.
|
|
| 9. |
- Li, Chunxia, et al.
(författare)
-
Noncovalent microarrays from synthetic amino-terminating glycans : Implications in expanding glycan microarray diversity and platform comparison
- 2021
-
Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:8, s. 931-946
-
Tidskriftsartikel (refereegranskat)abstract
- Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward beta-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.
|
|
| 10. |
- Li, Xiangyu, et al.
(författare)
-
Discovery of Functional Alternatively Spliced PKM Transcripts in Human Cancers
- 2021
-
Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:2, s. 1-23
-
Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Pyruvate kinase muscle type (PKM) is a key enzyme in glycolysis and is a mediator of the Warburg effect in tumors. The association of PKM with survival of cancer patients is controversial. In this study, we investigated the associations of the alternatively spliced transcripts of PKM with cancer patients' survival outcomes and explained the conflicts in previous studies. We discovered three poorly studied alternatively spliced PKM transcripts that exhibited opposite prognostic indications in different human cancers based on integrative systems analysis. We also detected their protein products and explored their potential biological functions based on in-vitro experiments. Our analysis demonstrated that alternatively spliced transcripts of not only PKM but also other genes should be considered in cancer studies, since it may enable the discovery and targeting of the right protein product for development of the efficient treatment strategies. Pyruvate kinase muscle type (PKM) is a key enzyme in glycolysis and plays an important oncological role in cancer. However, the association of PKM expression and the survival outcome of patients with different cancers is controversial. We employed systems biology methods to reveal prognostic value and potential biological functions of PKM transcripts in different human cancers. Protein products of transcripts were shown and detected by western blot and mass spectrometry analysis. We focused on different transcripts of PKM and investigated the associations between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcripts encoding PKM2 and three previously unstudied transcripts, namely ENST00000389093, ENST00000568883, and ENST00000561609, exhibited opposite prognostic indications in different cancers. Moreover, we validated the prognostic effect of these transcripts in an independent kidney cancer cohort. Finally, we revealed that ENST00000389093 and ENST00000568883 possess pyruvate kinase enzymatic activity and may have functional roles in metabolism, cell invasion, and hypoxia response in cancer cells. Our study provided a potential explanation to the controversial prognostic indication of PKM, and could invoke future studies focusing on revealing the biological and oncological roles of these alternative spliced variants of PKM.
|
|
