Quantum mechanical studies of residue-specific hydrophobic interactions in p53-MDM2 binding

Quantum chemistry calculations at the levels of MP2/cc-pVDZ and MP2/cc-PVTZ have been carried out to study residue-specific interactions at the hydrophobic p53-MDM2 binding interface. The result of the calculation, based on structures from nanosecond molecular dynamics simulation, revealed that (19)Phe, (22)Leu, and (23)Trp of p53 have the strongest binding interaction with MDM2 followed by (26)Leu and (27)Pro. The specific residues of MDM2 that have dominant binding interactions with p53 are specifically identified to be (51)Lys, (54)Leu, (62)Met, (67)Tyr, (72)Gln, (94)Lys, (96)His, and (100)Tyr. The p53-MDM2 binding interaction is dominated by van der Waals interaction and to a lesser degree by electrostatic interaction. The MP2 results are in generally good agreement with those from the force field calculation while the DFT/B3LYP calculation failed to give attractive interaction energies for certain residue-residue interactions due to the lack of dispersion energy.     

采用电活性标记物N-(2-乙基-二茂铁)马来酰亚胺检测与表面固定的 一致性双链DNA特异性作用的p53蛋白质

建立了电化学检测表面固定捕获的野生型p53蛋白质的方法. 首先在金电极表面形成巯基化的单链DNA探针/己硫醇(HT)混合自组装膜, 随后巯基化的单链DNA探针与溶液中序列匹配的靶点DNA杂交, 所形成的一致性双链DNA捕获溶液中的野生型p53蛋白质. p53分子表面的半胱氨酸残基采用巯基特异性试剂N-(2-乙基-二茂铁)马来酰亚胺(Fc-Mi)进行衍生. 通过检测二茂铁的电化学信号来指示p53与一致性双链DNA之间的特异性相互作用. p53蛋白质与双链DNA的键合程度取决于双链DNA的序列. 该方法可检测的p53最低浓度为1.33 nmol•L－1.     

Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53

The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.     

Proteomimetic libraries: design, synthesis, and evaluation of p53-MDM2 interaction inhibitors

The p53-MDM2 interaction regulates p53-mediated cellular responses to DNA damage, and MDM2 is overexpressed in 7% of all cancers. Structure-based computational design was applied to this system to design libraries centered on a scaffold that projects side chain functionalities with distance and angular relationships equivalent to those seen in the MDM2 interacting motif of p53. A library of 173 such compounds was synthesized using solution phase parallel chemistry. The in vitro competitive ability of the compounds to block p53 peptide binding to MDM2 was determined using a fluorescence polarization competition assay. The most active compound bound with K(d) = 12 microM, and its binding was characterized by (15)N-(1)H HSQC NMR.     

A graph theoretical approach to the effect of mutation on the flexibility of the DNA binding domain of p53 protein

Tumor suppressor protein p53 becomes inactive due to mutation on its DNA binding core domain leading to misbehavior of this protein and preventing its interaction with DNA. In the present study, changes of the protein conformation by five hot spot mutations of T-p53C were assessed preventing the mutants wild-type (WT) behavior. While studies of this nature were undertaken both experimentally and theoretically, the focus is fundamentally on the effects of the mutation on the dynamics of the protein. Hence, the basic concept underlying this study is the change in flexibility or rigidity of the protein. It was found that stable variant T-p53C (PDB-ID: 1uol) that is structurally and functionally very close to wild-type p53 is the most rigid structure and each single carcinogenic mutation on it makes the structure more flexible. We hypothesize that these changes of the molecule’s flexibility disrupt the network of hydrogen bonds associated with the interaction of WT not only at interaction but in the internal structures of the mutants as well, which prevents them from interacting in the WT fashion loosing the anti-cancer properties of WT.     

Increased Photosensitivity in HL60 Cells Expressing Wild-Type p53

Loss of p53 function has been correlated with decreased sensitivity to chemotherapy and radiation therapy in a variety of human tumors. Comparable analysis of p53 status with sensitivity to oxidative stress induced by pho-todynamic therapy has not been reported. In the current study we examined photosensitivity in human promye-locytic leukemia HL60 cells exhibiting either wild-type p53, mutated p53 or deleted p53 expression. Experiments were performed using a purpurin, tin ethyl etiopurpurin (SnET2)-, or a porphyrin, Photofrin (PH)-based photo-sensitizer. Total SnET2 accumulation was comparable in all three cell lines. Uptake of PH was highest in cells expressing wild-type p53 but incubation conditions could be adjusted to achieve equivalent cellular PH levels during experiments that analyzed photosensitivity. Survival measurements demonstrated that HL60 cells expressing wild-type p53 were more sensitive to PH- and SnET2-mediated photosensitization, as well as to UVC irradiation, when compared to HL60 cells exhibiting deleted or mutated p53 phenotypes. A rapid apoptotic response was observed following purpurin- and porphyrin-induced photosensitization in all cell lines. Results of this study indicate that photosensitivity is increased in HL60 cells expressing wild-type p53 and that photosensitizer-medi-ated oxidative stress can induce apoptosis through a p53-independent mechanism in HL60 cells .     

An integrated in silico screening strategy for identifying promising disruptors of p53-MDM2 interaction

The p53 protein, also called guardian of the genome, plays a critical role in the cell cycle regulation and apoptosis. This protein is frequently inactivated in several types of human cancer by abnormally high levels of its negative regulator, mouse double minute 2 (MDM2). As a result, restoration of p53 function by inhibiting p53-MDM2 protein–protein interaction has been pursued as a compelling strategy for cancer therapy. To date, a limited number of small-molecules have been reported as effective p53−MDM2 inhibitors. X-ray structures of MDM2 in complex with some ligands are available in Protein Data Bank and herein, these data have been exploited to efficiently identify new p53-MDM2 interaction antagonists through a hierarchical virtual screening strategy. For this purpose, the first step was aimed at compiling a focused library of 686,630 structurally suitable compounds, from PubChem database, similar to two known effective inhibitors, Nutlin-3a and DP222669. These compounds were subjected to the subsequent structure-based approaches (quantum polarized ligand docking and molecular dynamics simulation) to select potential compounds with highest binding affinity for MDM2 protein. Additionally, ligand binding energy, ADMET properties and PAINS analysis were also considered as filtering criteria for selecting the most promising drug-like molecules. On the basis of these analyses, three top-ranked hit molecules, CID_118439641, CID_60452010 and CID_3106907, were found to have acceptable pharmacokinetics properties along with superior in silico inhibitory ability towards the p53-MDM2 interaction compared to known inhibitors. Molecular docking and molecular dynamics results well confirmed the interactions of the final selected compounds with critical residues within p53 binding site on the MDM2 hydrophobic clefts with satisfactory thermodynamics stability. Consequently, the new final scaffolds identified by the presented computational approach could offer a set of guidelines for designing promising anti-cancer agents targeting p53-MDM2 interaction.     

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.

A hierarchical computational strategy for IDP drug virtual screening (IDPDVS) was proposed and successfully applied to identify compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells.     

Molecular dynamics simulation of S100B protein to explore ligand blockage of the interaction with p53 protein

As a tumor suppressor, p53 plays an important role in cancer suppression. The biological function of p53 as a tumor suppressor is disabled when it binds to S100B. Developing the ligands to block the S100B-p53 interaction has been proposed as one of the most important approaches to the development of anti-cancer agents. We screened a small compound library against the binding interface of S100B and p53 to identify potential compounds to interfere with the interaction. The ligand-binding effect on the S100B-p53 interaction was explored by molecular dynamics at the atomic level. The results show that the ligand bound between S100B and p53 propels the two proteins apart by about 2 Å compared to the unligated S100B-p53 complex. The binding affinity of S100B and p53 decreases by ~8.5–14.6 kcal/mol after a ligand binds to the interface from the original unligated state of the S100B-p53 complex. Ligand-binding interferes with the interaction of S100B and p53. Such interference could impact the association of S100B and p53, which would free more p53 protein from the pairing with S100B and restore the biological function of p53 as a tumor suppressor. The analysis of the binding mode and ligand structural features would facilitate our effort to identify and design ligands to block S100B-p53 interaction effectively. The results from the work suggest that developing ligands targeting the interface of S100B and p53 could be a promising approach to recover the normal function of p53 as a tumor suppressor.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

光谱法研究金属离子与肿瘤抑制蛋白p53的DNA结合结构域的相互作用

利用荧光滴定法研究了9种金属离子与序列特异性的DNA结合结构域(p53DBD)的结合反应,其结合能力依次为Fe3+＞Zn2+＞Cu2+＞Ca2+＞Mg2+＞Ba2+＞Mn2+＞Ni2+＞Co2+.圆二色谱研究结果表明,Ba2+, Ca2+, Co2+, Mn2+及Ni2+并未引起蛋白二级结构变化; Zn2+, Mg2+及Fe3+诱导蛋白结构细微调整;而Cu2+结合导致蛋白螺旋结构大量丢失.ANS结合研究结果表明, Mg2+与Zn2+相似,诱导p53DBD蛋白表面疏水性增强,而Fe3+引起p53DBD蛋白表面疏水性降低.因此,Mg2+和Fe3+可能是影响或调节p53活性的潜在因子之一.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

The p53 network: p53 and its downstream genes

The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network. p53 is a key molecular node in the network, which is activated in response to several cellular signals resulting in the maintenance of genetic stability. Several cellular signals may activate the p53 network. When the expression of P53 is elevated, P53-MDM2 module and the ubiquitin system can accurately regulate the expression level of P53. P53 can bind to specific DNA sequence, activate its downstream genes expression, and control cell-cycle arrest, DNA repair, and apoptosis. Elucidating the function of p53 gene network will help understand the interaction mechanisms of p53 and its downstream genes.     

Detection of mutant p53 using field-effect transistor biosensor

We assessed the abilities of wild p53 and mutant p53 proteins to interact with the consensus DNA-binding sequence using a MOSFET biosensor. This is the first report in which mutant p53 has been detected on the basis of DNA-protein interaction using a FET-type biosensor. In an effort to evaluate the performance of this protocol, we constructed the core domain of wild p53 and mutant p53 (R248W), which is DNA-binding-defective. After the immobilization of the cognate DNA to the sensing layer, wild p53 and mutant p53 were applied to the DNA-coated gate surface, and subsequently analyzed using a semiconductor analyzer. As a consequence, a significant up-shift in drain current was noted in response to wild p53, but not mutant p53, thereby indicating that sequence-specific DNA-protein interactions could be successfully monitored using a field-effect-based biosensor. These data also corresponded to the results obtained using surface plasmon resonance (SPR) measurements. Taken together, our results show that a FET-type biosensor might be promising for the monitoring of mutant p53 on the basis of its DNA-binding activity, providing us with very valuable insights into the monitoring for diseases, particularly those associated with DNA-protein binding events.