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Computer aided ligand design


Mutations in the p53 tumor suppressor gene frequently result in expression of p53 point mutants that accumulate in cancer cells and actively collaborate with tumor progression through the acquisition of novel properties. Interfering with mutant p53 functions may represent a valid alternative for blocking tumor growth and development of aggressive phenotypes. The interactions and activities of selected proteins can be specifically modulated by the binding of peptide aptamers (PA). We have isolated PAs able to interact more efficiently with p53 conformational mutants compared with wild-type p53. The interaction between mutant p53 and PAs was further characterized using molecular modeling. Transient expression of PAs was able to reduce the trans- activation activity of mutant p53 and to induce apoptosis specifically in cells expressing mutant p53. These PAs could provide a potential strategy to inhibit the oncogenic functions of mutant p53 and improve mutant p53-targeted cancer therapies.


Granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). In collaboration with Prof. Barth’s group at the Fraunhofer Institute at RWTH- Aachen, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications.

hSB9-hGBStructure of the hSB9 – hGB complex including the R201K mutation (yellow sticks)

Human angiogenin (hAng, also known as ribonuclease 5) is another potential immuntoxin for cancer therapy. hAng can eliminate tumor cells by cleaving the cellular RNA. Similar to hGB, the major limitation of using hAng as immunotoxin is the natural inhibitor, human placental RNase inhibitor (hRNH1), present in the cells. Based on the known structure of hAng in complex with hRNH1 (Fig. 2), we apply the same approach as that used for hGB to design hAng mutants with low binding affinity for hRNH1 without compromising the catalytic activity of the enzyme. Another limitation of hAng is its relatively low catalytic activity. To improve the catalytic efficiency, we study the enzymatic reaction of RNA cleavage by hybrid QM/MM simulations of the hAng-substrate (RNA) complex combined with metadynamics to investigate the free energy barrier of the reaction. The findings will enable to design hAng mutants with enhanced catalytic efficiency. This project is also in collaboration with Prof. Barth’s group at the Fraunhofer Institute.

hAng-hRNH1X-ray crystal structure of hAng-hRNH1 complex