Discovery of catechol-O-methyltransferase inhibitors through virtual screening

Abstract

BACKGROUND AND RATIONALE: Parkinson’s disease (PD) is a progressive neurodegenerative disease that is caused by the death of dopaminergic neurons in the substantia nigra resulting in a loss of dopamine in the striatum. Neurodegeneration in PD is typified by symptoms such as rigidity, tremor at rest, slowness (bradykinesia) and impairment of postural balance. Currently, there is no cure for PD and current therapies only provide symptomatic relief. In spite of several side effects, levodopa is still used in most cases, while several enzymes and receptors serve as drug targets. One of these targets is the monoamine oxidase (MAO) enzyme, in particular the MAO-B isoform. The MAO enzymes are responsible for the metabolism of amine neurotransmitters, such as dopamine. The inhibition of MAO-B has proven to be an effective strategy to increase dopamine levels in the brain. Since MAO-A is responsible for the breakdown of noradrenalin, adrenalin, serotonin and tyramine, non-selective and selective MAO-A inhibitors have therapeutic applications in other neurological and psychiatric disorders such as depression. MAO-A inhibitors, particularly irreversible inhibitors, are also notable from a toxicological point of view. Irreversible MAO-A inhibitors may lead to potentially dangerous effects when combined with serotonergic drugs and certain foods containing tyramine, such as cheeses and processed meats. Selective MAO-B inhibitors and reversible MAO-A inhibitors appear to be free of these interactions. The catechol-O-methyltransferase (COMT) enzyme is another enzymatic target. The inhibition of COMT results in a decrease of the clearance of L-dopa and dopamine, thus leading to a maintained level of dopamine in the brain and increased L-dopa efficacy. Currently used COMT inhibitors include tolcapone and entacapone. However, due to the side effects, which may include severe dopaminergic, gastrointestinal and other adverse reactions, their use is rather limited. Based on the considerations above, this study aimed to identify compounds with COMT inhibitory activity by virtual screening. The secondary aim of this study was to screen the same set of compounds for MAO inhibitory activity as the identification of a dual targeted compound would be an added advantage. METHODS: The following methods were used: Virtual screening: Firstly, three pharmacophore models were constructed using a crystal structure (PDB: 3BWM) of COMT. The Discovery Studio® software package (Accelrys) was used for this purpose. A virtual library of drugs approved by the United States Food and Drug Administration (FDA) were then screened. Secondly, in order to maximise the potential hits in this study, several other methods for identifying hits were used. These included the use of ligand fingerprinting, the use of molecular docking, the identification of catechol bioisosteres and compounds structurally related to known inhibitors such as kaempferol. In vitro screening: COMT inhibition was determined using a fluorometric assay and norepinephrine as substrate, while MAO inhibition was determined using a fluorometric assay and kynuramine as substrate. RESULTS: COMT inhibition studies: A list of twenty-six compounds were selected based on results from the pharmacophore mapping, screening of a library by fingerprinting, molecular docking, the bio-isostere approach, chemical similarity, cost and availability. These compounds were to be subjected to in vitro bio-assays (using porcine COMT) in order to determine their potencies (IC50 values) as inhibitors of COMT. Unfortunately, the Department of Fishery and Forestry placed a moratorium on the import of porcine products, which meant that the porcine COMT enzyme could no longer be obtained. The possibility of using the human enzyme was also investigated, but due to cost contraints its use was deemed unfeasible. Only eleven of the test compounds were thus evaluated as in vitro inhibitors of COMT. Among the compounds that were tested, only kaempferol (IC50 = 2.799 μM) exhibited inhibitory activity towards the COMT enzyme, most likely due to its structural similarity to quercetin. MAO inhibition studies: The IC50 values and selectivity index (SI) of eighteen compounds from the original COMT hit-list were also determined to investigate the inhibitory activity of these compounds towards an alternative target. Three of the eighteen test compounds exhibited promising IC50 values, and may thus be considered as MAO-A and MAO-B inhibitors. Kaempferol was the most potent MAOA inhibitor with an IC50 value of 0.589 μM and oxybenzone was the most potent MAO-B inhibitor with IC50 values of 24.967 μM and 2.872 μM for MAO-A and MAOB, respectively. Nitrendipine (16.353 μM) and (-)-riboflavin (13.119 μM) also showed some inhibition activity towards MAO-B. Docking studies: To complete this study and rationalise the results of the MAO inhibition studies, molecular modelling was carried out and the eighteen compounds screened for MAO-inhibitory activity were docked into the active sites of MAO-A and MAO-B by using the CDOCKER module of Discovery Studio®. Some insights were obtained regarding the binding of kaempferol, oxybenzone, nitrendipine and (-)- riboflavin. Both kaempferol and oxybenzone had hydrogen bond interactions with Cys 323, present in the active site of MAO-A. Thus, it may be concluded that a hydrogen bond interaction with Cys 323 may be an important feature for MAO-A inhibitory activity since clorgyline (a known MAO-A inhibitor) also undergoes this interaction. Furthermore, oxybenzone, the most potent MAO-B test inhibitor, successfully docked into the active site of MAO-B, although it did not illustrate hydrogen bond interactions with any of the nearby amino acid residues. Thus, it may be postulated that the binding of oxybenzone to the active site may be due to Van der Waals interactions with the amino acid residues. Furthermore, oxybenzone also share structural similarities with chalcones which has MAO inhibitory activity. The docking results for MAO-B also showed that most of the test compounds interacted with Tyr 326 or Tyr 398, while interactions with Cys 172, Gln 206, Ile 199 and Tyr 435 also occurred. Reversibility studies: To determine the reversibility of binding to MAO-B, the recovery of enzymatic activity after dialysis of enzyme-inhibitor complexes were determined for oxybenzone. The results indicated that the most potent MAO-B inhibitor, oxybenzone, had a reversible mode of binding to the MAO-B isoform, since the enzyme activity was completely recovered by dialysis. Mode of inhibition: To determine the mode of inhibition of oxybenzone, Lineweaver- Burk plots were constructed for the inhibition of MAO-B. The lines of the Lineweaver- Burk plots intersected at a single point at the y-axis, indicating that oxybenzone had a competitive mode of binding to the MAO-B isoform. The results of this study showed that virtual screening may be useful in identifying existing compounds with potential dual COMT and MAO inhibitory effects. In this study, for example, the dual inhibitory of both COMT and MAO by kaempferol was illustrated for the first time. Such an approach may also be more cost effective than the de novo design of COMT and MAO inhibitors