قادر حسین زاده

• Introduction Because vitamin B12 is a cofactor for enzymes involved in folate recycling for DNA synthesis, the need for B12 is increased in highly proliferative cells, including cancer cells [1]. In order to effectively absorb vitamin B12, the expression of CD320 gene increases in order to increase the production of TCblR protein (TC cell receptor bound to B12) [2]. For this reason, the TC/TCblR-mediated B12 uptake pathway has become an attractive potential target and as a marker for the design of anticancer drugs and therapeutic agents [3, 4]. Protein engineering is one of the new technologies in the field of designing new generation drugs based on proteins, which has attracted the attention of many researchers in recent years. Nanobodies (Nb), which are heavy and variable chains of specific antibodies of some animal species such as camels, are mainly used in the design of medicinal proteins due to their small structure and high stability [4, 5]. • Experimental / Methods In this regard, the aim of this work is to increase the binding affinity of engineered Nanobodies through designed mutations to blocking of TCblR cellular receptor by MD simulations. The all atom MD simulations were carried out using the GROMACS 5.1.2 MD package with GROMOS96 43a1 force field. The complex of TC with Nb (PDB: 7QBG) was used for MD simulations. • Results and discussion In MD simulations, by investigation the contact area and interactions between TC and Nanobody with COCOMAPS analysis, the residues of Nanobody with electrostatic repulsion interactions were identified, and based on these interactions, two mutations of ARG130 to GLU and ARG130 to ASP were proposed which labelled as V1 and V2 variants, respectively in order to improve the binding affinity of nanobody. • Conclusion According to the obtained results the mutation of ARG130 to GLU in V1 has the best binding affinity enhancement. The results of this work could be useful in designing of high affinity Nb against TCblR recepto