Chemical synthesis of transactivation domain (TAD) of tumor suppressor protein p53 by native chemical ligation of three peptide segments

Chemical composition of tumor suppressor protein p53 is altered via multiple post-translational modifications which modulate its cellular lifetime and interactions with other biomolecules. Here we report total chemical synthesis of a 61-residue form of transactivation domain (TAD) of p53 based on native chemical ligation of three peptide segments. The experiments to characterize its binding to nuclear co-activator binding domain (NCBD) of CREB-binding protein confirmed native-like induced folding upon binding to NCBD. Thus, the synthetic approach described herein can be useful for the preparation of various post-translationally modified analogues of TAD-p53 for further functional biochemical and biophysical studies.     

Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

Protein–protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.

Using aerolysin nanopores, we probed protein–protein interaction (PPI) between p53TAD and MDM2 and its modulation by small-molecule PPI inhibitors and p53TAD phosphorylation.     

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.     

Structure-based design of potent non-peptide MDM2 inhibitors

A successful structure-based design of a class of non-peptide small-molecule MDM2 inhibitors targeting the p53-MDM2 protein-protein interaction is reported. The most potent compound 1d binds to MDM2 protein with a Ki value of 86 nM and is 18 times more potent than a natural p53 peptide (residues 16-27). Compound 1d is potent in inhibition of cell growth in LNCaP prostate cancer cells with wild-type p53 and shows only a weak activity in PC-3 prostate cancer cells with a deleted p53. Importantly, 1d has a minimal toxicity to normal prostate epithelial cells. Our studies provide a convincing example that structure-based strategy can be employed to design highly potent, non-peptide, cell-permeable, small-molecule inhibitors to target protein-protein interaction, which remains a very challenging area in chemical biology and drug design.     

Harnessing Aromatic-Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high-resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor-1, which is targeted by osteoporosis drugs. These results should motivate other structure-based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.     

A Thiol–Ene Coupling Approach to Native Peptide Stapling and Macrocyclization

We report the discovery of a peptide stapling and macrocyclization method using thiol–ene reactions between two cysteine residues and an α,ω‐diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53‐MDM2 interaction and selectively kill p53 wild‐type colorectal carcinoma HCT‐116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides.     

Probing the Small‐Molecule Inhibition of an Anticancer Therapeutic Protein‐Protein Interaction Using a Solid‐State Nanopore

Nanopore sensing is an emerging technology for the single‐molecule‐based detection of various biomolecules. In this study, we probed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with a small‐molecule MDM2 antagonist, Nutlin‐3, using low‐noise solid‐state nanopores. Although the translocation of positively charged MDM2 through a nanopore was detected at the applied negative voltage, this MDM2 translocation was almost completely blocked upon formation of the MDM2/GST‐p53TAD complex owing to charge conversion. In combination with NMR data, the nanopore measurements showed that the addition of Nutlin‐3 rescued MDM2 translocation, indicating that Nutlin‐3 disrupted the MDM2/GST‐p53TAD complex, thereby releasing MDM2. Taken together, our results reveal that solid‐state nanopores can be a valuable platform for the ultrasensitive, picomole‐scale screening of small‐molecule drugs against protein–protein interaction (PPI) targets.     

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.     

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.     

SNARE derived peptide mimic inducing membrane fusion

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays.     

Targeting the conformational transitions of MDM2 and MDMX: insights into dissimilarities and similarities of p53 recognition

MDM2 and MDMX are oncogenic homologue proteins that regulate the activity and stability of p53, a tumor suppressor protein involved in more than 50% of human cancers. While the large body of experiments so far accumulated has validated MDM2 as a therapeutically important target for the development of anticancer drugs, it is only recently that MDMX has also become an attractive target for the treatment of tumor cells expressing wild type p53. The availability of structural information of the N-terminal domain of MDM2 in complex with p53-derived peptides and inhibitors, and the very recent disclosure of the crystal structure of the N-terminal domain of MDMX bound to a p53 peptide, offer an unprecedented opportunity to provide insight into the molecular basis of p53 recognition and the identification of discriminating features affecting the binding of the tumor suppressor protein at MDM2 and MDMX. By using coarse graining simulations, in this study we report the exploration of the conformational transitions featured in the pathway leading from the apo-MDM2 and apo-MDMX states to the p53-bound MDM2 and p53-bound MDMX states, respectively. The results have enabled us to identify a pool of diverse conformational states of the oncogenic proteins that affect the binding of p53 and the presence of conserved and non-conserved interactions along the conformational transition pathway that may be exploited in the design of selective and dual modulators of MDM2 and MDMX activity.     

Homology-free prediction of functional class of proteins and peptides by support vector machines

Protein and peptide sequences contain clues for functional prediction. A challenge is to predict sequences that show low or no homology to proteins or peptides of known function. A machine learning method, support vector machines (SVM), has recently been explored for predicting functional class of proteins and peptides from sequence-derived properties irrespective of sequence similarity, which has shown impressive performance for predicting a wide range of protein and peptide classes including certain low- and non- homologous sequences. This method serves as a new and valuable addition to complement the extensively-used alignment-based, clustering-based, and structure-based functional prediction methods. This article evaluates the strategies, current progresses, reported prediction performances, available software tools, and underlying difficulties in using SVM for predicting the functional class of proteins and peptides.