Constant pH simulations of Miglustat as a part of a multivalent ligand in GROMACSPotapov, Mark (Avtor) Podlipnik, Črtomir (Mentor) Lukšič, Miha (Komentor) MiglustatMultivalentcyConstant-pH simulationsGROMACS.This work is devoted to molecular simulations of protein and it is ligand at constant pH. The ligand is Miglustat (N-butyl-deoxynojirimycin) is the approved chemical compound for treatment of rare autosomal recessive diseases such as Gaucher and Pompe glycogenoses. One of the interesting applications of miglustat is its use as an inhitope in a multivalent drug. A multivalent compound consists of multiple copies of ligands that are typically attached by a flexible linker to a backbone structure. As a result, noncovalent interactions of multiple inhitope molecules with numerous binding sites of the receptor molecule(s) can be obtained. It was shown experimentally that reducing pH from 5.5 to 4 reduces the inhibition potency of 12 valent DNJ- based ligand by 400 times, while for a 4 valent ligand inhibition potency was reduced by 80 times . Recently, the thermodynamic non-cooperative model of multivalent binding was proposed. To complete this model with experimental observations, it was proposed to consider the contribution of electrostatic interactions between protein and ligand and different pH values. The charge of a molecule can be obtained from constant pH MD simulations. In 2022 the test version of the constant pH module of GROMACS was introduced (https://gitlab.com/gromacs- constantph/constantph). The idea is to test this code by calculating pH-dependent binding free energy of miglustat-based multivalent ligand. Recommended procedures of constant pH tutorial were followed, but because of encountered difficulties in-house protocols were developed, which allowed to set up and to simulate protein and ligand. As a result, charge of the protein at pH=5 is in reasonable agreement with experimental observations. Because the ligand is not a part of the titratable residues of the module, a parametrization procedure was successfully performed for it. Now it can be used to obtain the charge at specified pH value. The next steps could be: setting-up of the protein-ligand complex for constant-pH simulations, constant-pH simulations of the ligand and protein and different pH values.20252025-05-13 08:20:07Magistrsko delo/naloga169110sl