Abstract
In this dissertation, the ability of small molecules to influence protein-protein interactions (PPIs) and protein conformations are studied from a computational point of view. Various proteins are involved in diseases through their PPIs. Modulation of their PPIs can be employed in therapeutic applications and various existing drugs function via modulation of PPIs. Here, the influence of the molecular tweezer (CLR01) is assessed in the context of amyloidogenic proteins amyloid-beta (Aβ42), huntingtin and the human islet amyloid precursor protein (IAPP), as well as on the PPIs of a hub protein, 14-3-3ζ. For this purpose, we use computational methods such as molecular dynamics (MD) simulations, quantum mechanical/molecular mechanics (QM/MM) calculations and free energy simulations.
The aggregated form of Aβ42 peptide is believed to be involved in the pathology of Alzheimer’s disease. Two lysine residues of Aβ42 are considered to be important for its aggregation. Since CLR01 is able to selectively bind lysine residues, we employ replica exchange molecular dynamics (REMD) simulations to assess the effect of CLR01 binding to the Aβ42 dimer, the smallest toxic form of Aβ42 involved in the disease. We demonstrate that CLR01 encapsulates the two lysine residues and, to a lower extent, also the arginine residue of each Aβ42 monomer. This encapsulation leads to the disruption of the non-bonded interactions involving these residues, which destabilizes the Aβ42 dimer. Encapsulation of lysine and arginine is suggested to be important for the destabilizing effect as Aβ42 dimer formation is observed in similar REMD simulations with CLR03, the control molecule of CLR01 that does not form inclusion complexes.
The aggregation of the first exon of the huntingtin protein (htt exon-1) is involved in Huntington’s disease. Experimental data indicate that the first 17 residues of htt exon-1 (Nt17), which includes three lysine residues, are important for the aggregation. We hypothesize that binding of CLR01 to these residues is able to affect the aggregation and employed MD simulations, REMD simulations and QM/MM calculations to assess the effect of binding on htt exon-1 monomer structure. We demonstrate that CLR01 binding leads Nt17 to adopt a conformation with decreased α-helicity and weakened amphiphilic nature. The amphipathic helical nature of Nt17 is considered important for htt-exon-1 aggregation and loss of it might lead to a decreased aggregation.
The aggregation of hIAPP, a 37-residue long peptide, is linked to type-II diabetes. The lysine and arginine residues of hIAPP are important for its interactions with membranes and affect its conformation. CLR01 binding to the lysine residue of hIAPP is studied here using REMD simulations. CLR01 binding is found to stabilize a “kinked” form of hIAPP, rather than the aggregation-prone extended α-helix form. The conformational changes are accompanied with decrease in α-helicity, which could lower the toxicity.
CLR01 is also observed to stabilize the PPIs between 14-3-3ζ and Cdc25C peptide. This modulation of PPIs of 14-3-3ζ-Cdc25C by CLR01 is important for cell cycle regulation. Employing MD simulations and QM/MM calculations, we observe that CLR01 binds to an arginine residue of Cdc25C and fills the gap between the partner proteins at the interface, which stabilizes the PPI. Binding affinities of CLR01 to selective lysine and arginine residues of 14-3-3ζ and Cdc25C as well as the free energy associated with the PPI stabilization in presence of CLR01 are also computed using free energy simulations.
Another small molecule ME118, was observed to bind at the 14-3-3ζ dimerization interface. ME118 is the first small molecule that binds at the interface and modulates 14-3-3ζ PPIs. A combination of molecular docking and QM/MM MD simulations demonstrate that this is related to the higher amount of salt bridges and hydrogen bonding interactions at the interface than at other possible binding sites.
Overall, the results of this thesis provide insights into the inclusion complexes of CLR01 with lysine or arginine residues form different proteins and provide support for the role of CLR01 as a possible therapeutic for diseases associated with these proteins.! Structural information obtained for ME118 can be used for further development of molecules that might influence the dimerization behavior between different 14-3-3 isoforms.
