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Theoretical Investigations on the Interactions of Small Compounds with their Molecular Environments
(2015)
In the first part of this work, a combination of theoretical methods for the rational design of covalent inhibitor is presented. Starting from the crystal structure of the covalent complex of a lead compound, quantum mechanical and QM/MM calculations were used to derive the exact geometry of the preceeding non-covalent enzyme inhibitor complex. The geometry of the latter mainly determines the reactivity of the inhibitor against its target enzyme concerning the formation of the covalent bond towards an active site residue. Therefore, this geometry was used as starting point for the optimization of the substitution pattern of the inhibitor such as to increase its binding affinity without loosing its ability to covalently bind to the target protein. The optimization of the chemical structure was supported by using docking procedures, which are best suited to estimate binding affinities that arise from the introduced changes. A screening of the novel substitution patterns resulted in a first generation of model compounds which were further tested for their reactivity against the target. Dynamic simulations on the novel compounds revealed that the orientation that compounds adopt within the active site are such that a covalent interaction with the enzyme is no longer possible. Hence, the chemical structure was further modified, including not only changes in the substituents but also within the core of the molecule. Docking experiments have been conducted to assure sufficiently high binding affinities and to obtain the most favored binding poses. Those have then again been used for dynamic simulations which resulted in structures, for which the bond formation process appeared feasible. A final series of QM/MM calculations considering various protonation states was computed to estimate the reaction energies for the covalent attachment of the inhibitor to the enzyme. The theoretical results indicate a reasonable high inhibition potency of the novel compounds.
The second part concentrates on the environmental influences on the electron density of an inhibitor molecule. Therefore, a vinylsulfone-based model compound was selected for which an experimental crystal structure for the pure compound as well as a theoretically determined enzyme-inhibitor complex have been available. To provide reference data for the larger systems, the conformational space of the isolated molecule was screened for favorable geometries which were later compared to those within the crystal and protein surrounding. The geometry of the crystal structure could readily be taken from the experimental data whereas calculations on the protein complex revealed four potential non-covalent complexes exhibiting different arrangements of the molecule within the active site of the protein as well as two possible protonation states of the catalytic dyad. Hence, all four protein complexes have been compared to the crystal structure of the molecule as well as against the more favorable geometries of the isolated molecule being determined within vacuum or aqueous surrounding. Whereas the molecule itself was found to adopt comparable geometries within all investigated environments, the interactions pattern between the crystal surrounding and the protein differed largely from each other. The favorable formation of dimers within the crystal has a strong stabilizing effect and explains the extraordinarily good quality of the crystal. Within the protein however, repulsive forces have been found between the protein and the inhibitor. The origin of the repulsion could be traced back to effect of on of the substituents to the vinyl scaffold. The difference in the chemical structure in comparison to a well known inhibitor might also explain the experimentally found loss of activity for the model compound in comparison to K11777.
Mechanistic Insights into the Inhibition of Cathepsin B and Rhodesain with Low-Molecular Inhibitors
(2019)
Cysteine proteases play a crucial role in medical chemistry concerning various fields reaching from more common ailments like cancer and hepatitis to less noted tropical diseases, namely the so-called African Sleeping Sickness (Human Arfican Trypanosomiasis). Detailed knowledge about the catalytic function of these systems is highly desirable for drug research in the respective areas. In this work, the inhibition mechanisms of the two cysteine proteases cathepsin B and rhodesain with respectively one low-molecular inhibitor class were investigated in detail, using computational methods. In order to sufficiently describe macromolecular systems, molecular mechanics based methods (MM) and quantum mechanical based method (QM), as well as hybrid methods (QM/MM) combining those two approaches, were applied.
For Cathespin B, carbamate-based molecules were investigated as potential inhibitors for the cysteine protease. The results indicate, that water-bridged proton-transfer reactions play a crucial role for the inhibition. The energetically most favoured pathway (according to the calculations) includes an elimination reaction following an E1cB mechanism with a subsequent carbamylation of the active site amino acid cysteine.
Nitroalkene derivatives were investigated as inhibitors for rhodesain. The investigation of structurally similar inhibitors showed, that even small steric differences can crucially influence the inhibition potential of the components. Furthermore, the impact of a fluorination of the nitroalkene inhibitors on the inhibition mechanism was investigated. According to experimental data measured from the working group of professor Schirmeister in Mainz, fluorinated nitroalkenes show – in contrast to the unfluorinated compounds – a time dependent inhibition efficiency. The calculations of the systems indicate, that the fluorination impacts the non-covalent interactions of the inhibitors with the enzymatic environment of the enzyme which results in a different inhibition behaviour.
The SARS virus is the etiological agent of the severe acute respiratory syndrome, a deadly disease that caused more than 700 causalities in 2003. One of its viral proteins, the SARS coronavirus main protease, is considered as a potential drug target and represents an important model system for other coronaviruses. Despite extensive knowledge about this enzyme, it still lacks an effective anti-viral drug. Furthermore, it possesses some unusual features related to its active-site region. This work gives atomistic insights into the SARS coronavirus main protease and tries to reveal mechanistic aspects that control catalysis and inhibition. Thereby, it applies state-of-the-art computational methods to develop models for this enzyme that are capable to reproduce and interpreting the experimental observations. The theoretical investigations are elaborated over four main fields that assess the accuracy of the used methods, and employ them to understand the function of the active-site region, the inhibition mechanism, and the ligand binding. The testing of different quantum chemical methods reveals that their performance depends partly on the employed model. This can be a gas phase description, a continuum solvent model, or a hybrid QM/MM approach. The latter represents the preferred method for the atomistic modeling of biochemical reactions. A benchmarking uncovers some serious problems for semi-empirical methods when applied in proton transfer reactions. To understand substrate cleavage and inhibition of SARS coronavirus main protease, proton transfer reactions between the Cys/His catalytic dyad are calculated. Results show that the switching between neutral and zwitterionic state plays a central role for both mechanisms. It is demonstrated that this electrostatic trigger is remarkably influenced by substrate binding. Whereas the occupation of the active-site by the substrate leads to a fostered zwitterion formation, the inhibitor binding does not mimic this effect for the employed example. The underlying reason is related to the coverage of the active-site by the ligand, which gives new implications for rational improvements of inhibitors. More detailed insights into reversible and irreversible inhibition are derived from in silico screenings for the class of Michael acceptors that follow a conjugated addition reaction. From the comparison of several substitution patterns it becomes obvious that different inhibitor warheads follow different mechanisms. Nevertheless, the initial formation of a zwitterionic catalytic dyad is found as a common precondition for all inhibition reactions. Finally, non-covalent inhibitor binding is investigated for the case of SARS coranavirus main protease in complex with the inhibitor TS174. A novel workflow is developed that includes an interplay between theory and experiment in terms of molecular dynamic simulation, tabu search, and X-ray structure refinement. The results show that inhibitor binding is possible for multiple poses and stereoisomers of TS174.