Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration

Abstract

HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to "dirty drugs" for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are simply symptomatic treatment so if new drugs are to prevent degeneration rather than compensate for loss of neurotransmitters, then oxidative stress and mitochondrial events must also be targeted. MAO inhibitors can protect neurons from apoptosis by mechanisms unrelated to enzyme inhibition. Understanding the involvement of MAO and other proteins in the induction and regulation of the apoptosis in mitochondria will aid progress toward strategies to prevent the loss of neurons. In general, the oxidative stress observed both in PD and AD indicate that antioxidant properties are a desirable part of MTDL molecules. After two or more properties are incorporated into one molecule, the passage from a lead compound to a therapeutic tool is strictly linked to its pharmacokinetic and toxicity. In this context the interaction of any new molecules with cytochrome P450 and other xenobiotic metabolic processes is a crucial point. The present review covers the biochemistry of enzymes targeted in the design of drugs against neurodegeneration and the cytochrome P450-dependent metabolism of MTDLs.

Keywords: cytochrome P450; mitochondria; monoamine oxidase; multi target designed ligands; neurodegeneration; oxidative stress.

Figure 1

Ligand binding cavities of (A) AChE and (B) BChE. AChE (shown in orange) is in complex with donepezil (shown in CPK colored sticks with carbons in green, PDB ID: 4EY7), while BChE (shown metal blue) is in complex with choline (shown in CPK colored sticks with carbons in light blue, PDB ID: 1P0M). The top panels show the cartoon representations with detail in sticks of relevant residues involved at the gorge entrances, the PAS regions or the catalytic triads (labeled in red), as well as the omega loops colored in yellow. Middle and lower panels show top and lateral views of the ligand binding cavities. The entrance loops are highlighted in pink and yellow respectively. PDB files were obtained from the protein databank and figures were produced using the PyMol software (PyMOL, http://www.pymol.org).

Figure 2

MAO active site cavities showing the FAD cofactor and the ligand in the crystal structures. (A) MAO A in complex with clorgyline (PDB ID: 2BXS) and (B) MAO B in complex with deprenyl (PDB ID: 2BYB). The FAD cofactors inside the cavities are shown in CPK colored sticks with carbons in pink, the clorgyline and deprenyl ligands are also shown in sticks with carbons in blue, and key residues around the ligand cavity are shown in CPK colored sticks with carbons in orange and metal blue for MAO A and MAO B, respectively. The entrance loops are highlighted in pink and yellow respectively. The PDB files were obtained from the protein databank and figures were produced using PyMOL (http://www.pymol.org).

Figure 3

Schematic representation of mitochondrial dynamics. Steady state mitochondrial morphology requires a balance of fission and fusion events. During organelle fission Drp1 is recruited from the cytosol to the outer mitochondrial membrane, where it interacts directly or indirectly with Fis1 forming high molecular weight oligomers on the mitochondrial surface. This leads to constriction of mitochondria and sequential separation of the inner and outer membrane. Once Drp1 is released fission is complete. Fission also allows isolation for mitochondria that cannot be repaired followed by degradation through mitophagy, and is also important for subcellular distribution and transportation of mitochondria based on local energy needs. Mitochondrial fusion is a two-step process that requires outer and inner membrane fusion. Outer membrane fusion is facilitated by mitofusins tethering of adjacent membranes. This is subsequently followed by inner membrane fusion, which is GTP dependent and regulated by OPA1. Fusion allows for functional complementation and repair of damaged mitochondria.

Figure 4

Schematic representation of the timeline of mitochondrial bioenergetics and morphological changes inducing pathologies. Electrons leaking from the electron transport chain generate ROS, which damage mitochondrial membrane, mitochondrial DNA, and proteins. Neurons have limited defense against oxidative damage and are highly vulnerable to ROS. Damaged/depolarized mitochondria release cytochrome c that triggers cell death by activating caspases as well as AIF that initiates apoptosis in a caspase independent manner.

Figure 5

Compound SMe1EC2 compared with the parent drug stobadine according to the structural, and in vitro and in vivo properties.

Figure 6

Cytochrome P450-dependent metabolism of ASS234 in human (HLM) and rat (RLM) liver microsomal preparations. ASS234 (25 μM) was incubated at 37°C in phosphate buffer in the presence of microsomes for 30 min.