Cancer

Sirtuin enzymes belong to the family of class III NAD⁺-dependent deacetylases that are conserved from bacteria to human. In mammals, seven sirtuins (SIRT1-SIRT7) have been identified which share a similar catalytic core. SIRT2 has recently emerged for its high therapeutic potentials in cancer and some other age-related diseases. Growing evidence suggests that inhibition of SIRT2 can gain function in cell fate and tumorigenesis through regulating acetylation status of several tumor suppressors including p53 and FoxO1. Despite the great interesting in SIRT2 inhibitor, the current SIRT2 inhibitors have severe limitations especially in selectivity. Small molecule inhibitors of SIRT2 targeting the catalytic core such as Sirtinol, can also inhibit or bind with other sirtuins. Recent in vitro experiments indicate that the phosphorylation of SIRT2 at C-terminal inhibits the deacetylation activity, which leads a promising way to design more effective and selective SIRT2 inhibitors. In collaboration with molecular biology and biochemistry group of Prof. B. Lüscher in RWTH Aachen-Clinics, we are investigating the inhibition mechanism regulated by SIRT2 phosphorylation using molecular modeling protocols.

Human Granzyme B-based agents represent promising anticancer strategies. Indeed, Granzyme B is a serine protease involved in cell-mediated apoptosis. However, its toxicity against cancer cells is limited by the expression of the inhibitor Serpin B9, another serine protease able to form an irreversible covalent complex with Granzyme B. In collaboration with Prof. Barth’s group at the Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME-MB in Aachen and RWTH-University of Aachen, we are designing potential Granzyme B mutants able to preserve their physiological activity while decreasing their binding to Serpin B9. In order to identify the crucial residues for the Granzyme B-Serpin B9 interactions, we have first modeled the Serpin B9 structure and its interaction with Granzyme B based on an analogous protein complex. Then, we have performed alanine scanning in order to define hot spot residues able to significantly decrease the Granzyme B-Serpin B9 binding free energy upon mutation to alanine. Variants of those residues have been designed and are currently being simulated in order to assess their destabilizing effects. The results will be then tested by mutagenesis and in vitro activity essays. The most promising variants will represent possible candidates for therapeutical applications.

The PARP (poly-ADP-ribose polymerases) family is a group of enzymes involved in mono- and poly-ADP-ribosylation. This event represents a crucial cellular response to DNA damage and it plays an important role in converting normal cells into a malignant state. PARP enzymes polyADP-ribosylate themselves and nuclear proteins, using nicotinamide adenine dinucleotide (NAD) as a substrate. In addition, they are involved in the control of transcription and cellular differentiation. Among these enzymes, the PARP10 protein is also able to interact with the proto-oncoprotein Myc, thus directly controlling tumor cell proliferation. However, unlike other members of its family, PARP10 lacks the catalytic glutamate (structurally substituted by an isoleucine) in the active site common "HYE" motif and it is able to perform only mono-ADP ribosylation. Therefore, a substrate-assisted catalytic mechanism was hypothesized, in which an acidic target residue of the substrate functionally substitutes for the missing glutamate. In collaboration with the group of Prof. Lüscher (RWTH Aachen Clinics), we are characterizing the interaction between PARP10 and its substrate GSK3B using protein-protein docking procedures. Elucidating the details of this interaction will help understanding the biological role of mono-ADP ribosylation.

Fig. 3. a) Model of the PARP10 (silver cartoon) - NAD⁺ (green sticks) complex. The catalytic residues are represented in orange sticks. b) Structure of the GSK3B protein (cyan cartoon). Yellow sticks represent the glutamates under investigation.

The anticancer effect of cisplatin was discovered by chance more than 40 years ago and since then it has made a major impact in cancer chemotherapy. Nowadays, it remains widely used to treat various types of cancer diseases such as testicular cancer, ovarian cancer, cervical cancer, colorectal cancer and relapsed lymphoma. Cisplatin destroys cancer cells by binding to the DNA, forming cisplatin-damaged DNA adducts, which distort the DNA structure. However, the development of resistance against cisplatin is one of the major challenges in cancer chemotherapy. Understanding cellular mechanism of cisplatin resistance may help design new drugs with better efficacy. It has been observed that in resistant cells, the cellular accumulation of cisplatin is reduced dramatically. This is intimately related to the altered activity of proteins involved in the cellular uptake and efflux of cisplatin. In this context, the high-affinity copper transporter Ctr1 has been found to mediate the uptake of cisplatin. whereas the copper chaperone Atox1 and copper pumps ATP7A/ATP7B to be involved in efflux/sequestration of cisplatin. We are working with the experimental group of Prof. Giovanni Natile at University of Bari, Italy and group of Prof. Luisa De Cola at University of Münster, Germany to characterize the structural and spectroscopic properties of cisplatin binding to these copper transport proteins. We are using a set of computational tools based on QM/MM simulations, force matching and replica exchange MD to investigate these fascinating aspects of cisplatin cell biology. This research is supported by a DFG grant.

Fig. 4. Model of cisplatin uptake mediated by Ctr1 (Adapted from Arnesano, F. et al. Angew. Chem. Int. Ed. Engl. 2007, 46, 9062).