Chemical Protein Synthesis – a Powerful Tool for Basic Research and Drug Discovery. Protein research has been reinvigorated in the post-genomic era, where innovative tools and enabling technologies for protein science play an essential role in basic and translational research. Chemical protein synthesis via native chemical ligation is one such tool/technology. Native chemical ligation allows for multiple small peptides to assemble into a large polypeptide chain, making it possible to chemically synthesize domain-sized proteins containing not only coded amino acids but also non-coded building blocks. Several ongoing projects at IHV showcase the power of chemical protein synthesis in contemporary peptide/protein research at the interface between chemistry and biology.
As a chemical engineer of proteins, Dr. Lu is interested in knowing what happens to structural and functional properties of proteins when their side chains and backbone are altered in a way that is genetically unattainable. His laboratory, in collaboration with Drs. Pazgier, de Leeuw and Garzino-Demo, has also applied the chemical protein engineering approach to decipher the molecular determinants of the diverse functions of human defensins in innate host defense and adaptive immunity, and to elucidate their mechanisms of action against infectious microbes. Such knowledge may help design new anti-infective agents to treat infectious diseases. More recently, his team, in collaboration with Dr. Pazgier and others, has discovered, by combining chemical protein synthesis, phage display and rational design, several peptide antagonists of the oncogenic protein MDM2 that selectively kill tumor cells by reactivating the p53 tumor suppressor. A parallel study is underway that aims to develop D-peptide inhibitors of HIV assembly and maturation by targeting the matrix and capsid domains of the HIV Gag polyprotein. Since chemical protein synthesis affords facile access to proteins that are often difficult to produce recombinantly, permits regiospecific and controlled disulfide formation, and allows site-specific chemical labeling and post-translational modifications, this powerful tool can facilitate protein structure determination as well by X-ray crystallography and NMR spectroscopy.
Liu M, Li C, Pazgier M, Li C, Mao Y, Lv Y, Gu B, Wei G, Zhan C, Yuan W, Lu W-Y, Lu W (2010). D-peptide inhibitors of the p53-MDM2 interaction for targeted molecular therapy of malignant neoplasms. Proc Natl Acad Sci USA 107(32):14321-6.
Li C, Li X, Lu W (2010). Total chemical synthesis of human T-cell leukemia virus type 1 protease via native chemical ligation. Biopolymers 94(4):487-494.
Liu M, Pazgier M, Li C, Yuan W, Li C, Lu W (2010). A left-handed solution to peptide inhibition of the p53-MDM2 interaction. Angew Chem Int Ed Engl 49(21):3649-52.
Li C, Pazgier M, Li J, Li C, Liu M, Zou G, Li Z, Chen J, Tarasov SG, Lu W-Y, Lu W (2010). Limitations of peptide retro-inverso isomerization in molecular mimicry. J Biol Chem 285(25):19572-81.
Li C, Pazgier M, Li C, Yuan W, Liu M, Wei G, Lu WY, Lu W (2010). Systematic mutational analysis of peptide inhibition of the p53-MDM2/MDMX interactions. J Mol Biol 398(2):200-13.
Wei G, Pazgier M, de Leeuw E, Rajabi M, Li J, Zou G, Jung G, Yuan W, Lu WY, Lehrer RI, Lu W (2010). Trp-26 imparts functional versatility to human alpha-defensin HNP1. J Biol Chem 285(21):16275 85.
de Leeuw E, Li C, Zeng P, Li C, Buin MD, Lu WY, Breukink E, Lu W (2010). Functional interaction of human neutrophil peptide 1 with the cell wall precursor lipid II. FEBS Lett 584(8):1543-8.
Li C, Pazgier M, Liu M, Lu WY, Lu W (2009). Apamin as a template for structure-based rational design of potent peptide activators of p53. Angew Chem Int Ed Engl 48(46):8712-5.
Wei G, de Leeuw E, Pazgier M, Yuan W, Zou G, Wang J, Ericksen B, Lu WY, Lehrer RI, Lu W (2009). Through the looking glass, mechanistic insights from enantiomeric human defensins. J Biol Chem 284(42):29180-92.
Pazgier M, Liu M, Zou G, Yuan W, Li C, Li C, Li J, Monbo J, Zella D, Tarasov SG, Lu W (2009). Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX. Proc Natl Acad Sci USA 106(12):4665-70.
Figure Legend: The oncogenic protein MDM2 – an E3 ubiquitin ligase negatively regulates the activity and stability of the p53 tumor suppressor, a cellular event initiated by MDM2 sequestration of the N-terminal transactivation domain of p53. Inhibitors that disrupt the p53-MDM2 interaction activate the p53 pathway and induce p53-dependent killing of tumor cells in vitro and in vivo. By screening a phage-expressed duodecimal peptide library against a chemically synthesized, site-specifically biotinylated D-enantiomer of the p53-binding domain of MDM2, we have identified two distinct D-peptide antagonists of MDM2 with potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Shown in the figure are crystal structures of the two D-peptide inhibitors bound, in a left-handed helical conformation, to the p53-binding pocket of MDM2; the most critical residues for MDM2 recognition are depicted in ball and stick on the molecular surface of the protein.