Browsing by Subject "docking"

11β-hydroxysteroid dehydrogenase/reductase (11β-HSD) enzymes 1 and 2 regulate the amount of cortisone and cortisol in human tissues. Since overexpression of 11β-HSD1 especially in the adipose tissue causes symptoms of metabolic syndrome, selective inhibition of 11β-HSD1 provides a way to treat this syndrome and type II diabetes. Inhibition of 11β-HSD2 causes cortisol-dependent mineralocorticoid activation, which leads to hypertensive side effects. There are several reported 11β-HSD1 inhibitors, for selective 11β-HSD2 inhibitition, only a few compounds have been developed. The difference between 11β-HSD1 and 2 ligand binding sites is unknown, which complicates the search of selective inhibitors to both of the enzymes. This study was done with two aims: (1) to identify the difference between the two isozymes, (2) to create pharmacophore models for selective 11β-HSD2 inhibitiors. These tasks were approached with computational methods: homology modeling, docking, ligand-based pharmacophore modeling and virtual screening. The homology model of 11β-HSD2 was constructed using SwissModeler and it showed satisfying superimposition both with is template 17β-HSD1 and 11β-HSD1. The difference between the enzymes could not be identified by visual inspections Therefore, seven compounds, of which six are 11β-HSD2 -selective, were docked both to 11β-HSD1 and 11β-HSD2 ligand binding sites using the program GOLD. The docking results revealed that the compounds orientate differently in the enzymes. To 11β-HSD1, the compounds were anchored similar than unselective compound carbenoxolone, whereas in 11β-HDS2, they adopted a flipped binding mode. The flipped binding mode in 11β-HDS2 enables hydrogen bonds to Ser310 and to Asn171, both residues that are only present in 11β-HSD2. Pharmacophore modeling and virtual screening were done using the program LigandScout3.0. The ligand-based pharmacophores were based on the six 11β-HSD2 selective compounds, which were also used for the docking studies. Both of the models consisted of six features (hydrogen bond acceptors, hydrogen bond donor and hydrophobic feature) besides the exclusion volumes. The most important features considering the 11β-HSD2 selectivity seem to be the hydrogen bond acceptor feature that could interact with the Ser310 and the hydrogen bond donor feature next to it. The interaction pair for this hydrogen bond donor feature was not observed in the homology model. However, a possibility of water molecule as an interaction pair was evaluated and it seems to be a possible solution to the problem. Since both of the models were able to find the selective 11β-HSD2 inhibitors and exclude the unselective ones from the test set database, they were employed for the screening of the database that consists of 2700 compounds stored at the University of Innsbruck. From the hits of these screenings ten compounds were selected and sent to biological testing. The results of the biological tests will decide how well the models represent the theory of the 11β-HSD2 selectivity.

New drugs against malaria are required, as millions of people are still affected yearly by this deadly disease. The development of drug resistance to current antimalarials is an ongoing process. Membrane-bound pyrophosphatases (mPPases) are potential new drug targets against malaria and other protozoan diseases. mPPases play a crucial role in the survival of the malaria parasite, they couple the energy released from the hydrolysis of pyrophosphate into the transport of protons or ions against an electrochemical gradient. The aim of this study was to identify potential mPPase inhibitors through a docking-based virtual screen of the Tres Cantos Antimalarial Compound Set, which consists of over 13500 malaria-active compounds. The virtual screen against a Thermotoga maritima mPPase protein structure identified a 2,4-diamino-1,6-dihydrotriazine among the top-ranking scaffolds. Four compounds found among the docking results containing this scaffold were synthesised: three with a halophenyl substituent, and one with a hydroxyl substituent. The compounds in their hydrochloride salt forms were synthesised using a three-component method for the synthesis of 2,4-diamino-1,6-dihydrotriazines. The compounds were also freed from the hydrochloride salts into their corresponding molecular forms. The structural characterisation of the compounds, especially the molecular forms, presented challenges. The docking results were also searched to identify compounds containing previously identified mPPase-active substructures. From the docking results, several other interesting compounds were identified in addition to the synthesised compounds. The knowledge and results obtained from this study can be used as openings for potential future docking and synthesis projects in the development of mPPase inhibitors. The activity of the compounds synthesised in the project remains to be evaluated in subsequent investigations.

Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in maturating high-density lipoprotein (HDL) particles, has been targeted to promote the efficiency of reverse cholesterol transport by small molecular positive allosteric modulators (PAM) of Daiichi Sankyo. For a set of these compounds their Vmax and EC50 values and binding site in the membrane-binding domain (MBD) of LCAT have been determined. Through molecular dynamics (MD) simulations we previously found a metric that qualitatively described which compounds were active, so in this study we aimed to improve it by finding a quantitative metric. This led to the discovery of the Cα distance between CYS50 and ASN65, which correlates with this set’s Vmax values and which can be utilized to predict the Vmax values of novel compounds. Additional simulations were performed to discover whether this metric is changed by a lipid interface present, and to reveal a likely entry pathway PAMs take. As LCAT activation is likely a benign and potentially overlooked effect, we performed a virtual screen of FDA-approved compounds and secondary metabolites associated with LCAT. From secondary metabolites, a key finding was that flavonoids were overwhelmingly associated with LCAT and had a high binding potential to the MBD in docking simulations. The best binding compounds were subjected to MD simulations to discover their Vmax values using the discovered metric. This provided us with a set of compounds, which can be used to validate our in silico model in vitro. Should this model be validated, it can be used in optimising and discovering novel PAMs of LCAT, and it would bring evidence to the benefit of MD in drug discovery processes in general. Furthermore, if our discovered compounds can activate LCAT in vitro, they may be used as precursors for novel PAMs or as therapies by themselves not only for LCAT deficiencies, but perhaps for atherosclerotic cardiovascular diseases as well.

Cancer is a worldwide health problem; in 2018 9.6 million people died of cancer, meaning that about 1 in 6 deaths was caused by it. The challenge with cancer drug therapy has been the development of cancer drugs that are effective against cancer but are not harmful to the healthy cells. One of the solutions to this has been antibody-drug conjugates (ADCs), where a cytotoxic drug is bound to an antibody. The antibody binds to specific antigen present on the surface of the cancer cell, thus working as a vessel to carry the drug specifically to the cancer cells. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are mitosis preventing cancer drugs. The auristatins are pentapeptides that were developed from dolastatin 10. MMAE consist of monomethyl valine (MeVal), valine (Val), dolaisoleiune (Dil), dolaproine (Dap) and norephedrine (PPA). MMAF has otherwise similar structure, but norephedrine is replaced by phenylalanine (Phe). They prevent cell division and cancer cell proliferation by binding to microtubules and are thus able to kill any kind of cell. By attaching the auristatin to an antibody that targets cancer cells, they can effectively be used in the treatment of cancer. MMAE and MMAF exist as two conformers in solution, namely as cis- and trans-conformers. The trans-conformer resembles the biologically active conformer. It was recently noted that in solution 50-60 % of the MMAE and MMAF-molecules exist in the biologically inactive cis-conformer. The molecule changes from one conformer to the other by the rotation of an amide bond. However, this takes several hours in body temperature. As the amount of the cis-conformer is significant, the efficacy of the drug is decreased, and the possibility of side effects is increased. It is possible that the molecule leaves the cancer cell in its inactive form, migrates to healthy cells and tissue, and transforms to the active form there, damaging the healthy cell. The goal of this study was to modify the structure of the auristatins so that the cis/trans-equilibrium would change to favor the biologically active trans-conformer. The modifications were done virtually, and the relative energies were computed using high-level quantum chemical methods, at density functional theory (DFT), 2nd order perturbation theory (MP2) and coupled cluster levels. Intramolecular interactions were analyzed computationally, employing symmetry-adapted perturbation theory and the non-covalent interactions analysis. The results suggest that simple halogenation of the benzene ring para-position is able to significantly shift the cis/trans-equilibrium to favor the trans-conformer. This is due to changes in intramolecular interactions that favor the trans-conformer after halogenation. For example, the NCI analysis shows that the halogen atom invokes stabilizing intramolecular interactions with the Dil amino acid; there is no such interaction between the para-position hydrogen and Dil in the original molecules. We also performed docking studies that show that the halogenated molecules can bind to microtubules, thus confirming that the modified structures have potential to be developed into new, more efficient and safe cancer drugs. The most promising drug candidates are Cl-MMAF, F-MMAF, and F-MMAE where 94, 90, and 79 % of the molecule is predicted to exist in the biologically active trans-conformer, respectively.

Membrane-bound pyrophosphatases (mPPases) are a potential target for drugs against many neglected protozoan diseases, such as malaria, leishmaniasis, toxoplasmosis and trypanosomiasis. New drugs against these diseases are urgently needed, as the clinically used ones are either not effective, suffer from side effects, or resistance against them is developing. The mPPases of these protozoans are genetically conserved, while mammalian DNA does not encode them. A drug development project to find mPPase inhibitors was started, based on mPPase structures solved through X-Ray crystallography. Four hit compounds were identified. The aim of this study was to investigate the binding of these hit compounds at the mPPase binding site, and based on these results, to develop and synthesize novel compounds with higher affinity. A hit compound with an isoxazole ring was chosen as the model compound to be developed further. These novel compounds were evaluated by docking them into the binding site. Eight compounds were chosen to be synthesized and four to be purchased. The Suzuki-Miyaura cross-coupling reaction was used to couple the isoxazole core to different aromatic substituents, producing 3,5-disubstituted isoxazoles. The reactions mostly succeeded, but the yields were uniformly low. Developing the reaction using different solvents and reaction conditions did not produce clear results. Thirteen compounds were tested for activity, including an intermediate product of the synthesis. Two of the compounds showed increased inhibition activity compared to the hit compound, with approximated IC50 values of 10 and 40 μM, respectively. The knowledge gained from these studies can be used to further develop more efficient inhibitors.