Willardiine (correctly spelled with two successive i's) or (S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione is a chemical compound that occurs naturally in the seeds of Mariosousa willardiana and Acacia sensu lato. The seedlings of these plants contain enzymes capable of complex chemical substitutions that result in the formation of free amino acids (See: #Synthesis). Willardiine is frequently studied for its function in higher level plants. Additionally, many derivates of willardiine are researched for their potential in pharmaceutical development. Willardiine was discovered in 1959 by R. Gmelin, when he isolated several free, non-protein amino acids from Acacia willardiana (another name for Mariosousa willardiana) when he was studying how these families of plants synthesize uracilyalanines. A related compound, Isowillardiine, was concurrently isolated by a different group, and it was discovered that the two compounds had different structural and functional properties. Subsequent research on willardiine has focused on the functional significance of different substitutions at the nitrogen group and the development of analogs of willardiine with different pharmacokinetic properties. In general, Willardiine is one of the first compounds studied in which slight changes to molecular structure result in compounds with significantly different pharmacokinetic properties.
Willardiine is a partial agonist of Ionotropic glutamate receptors. These receptors are found at excitatory synapses and bind glutamate (the major excitatory neurotransmitter) and structurally similar ligands, such as willardiine. Receptor activation leads to influx of positive ions into the neuron, resulting in neural depolarization (See: #Structure and Activity). Willardiine specifically agonizes non-NMDA glutamate receptors: AMPA and kainate receptors.
Willardiine analogs have been developed that have different binding affinities for the AMPA and kainate receptors. These analogs have been used to study the structure of these receptors, as well as the functional significance of receptor activation in different brain regions. While willardiine and its analogs have not been explicitly studied as therapeutics, there are a variety of neurological disorders characterized by alterations in glutamate signaling, and ligands for AMPA and kainate receptors are often studied as potential therapeutics.
Chemistry
Structure and activity
Structure Willardiine was isolated from Acacia Willardiana and characterized as 3-(1-uracyl)--alanine, based on elemental composition and stability in strong acid, which were used to deduce the organic structure of willardiine (Figure 1). The experimentally derived properties of willardiine are similar to -albizziine, which was isolated from the same seed. Subsequent synthetic work proved the structure and properties of willardiine (see: #Synthesis).
The family of willardiine compounds (see: #Analogs) all have a uracil or substituted uracil as the primary amino acid side chain. Willardiine naturally exists as two isomers:
(S)-Willardiine (R)-Willardiine Only the (S) isomer has binding affinity for the AMPA and kainate receptors. Isomer-specific binding affinity is a result of steric effects between (R)-Willardiine and the binding site on the receptor.
Activity
AMPA receptors are tetrameric transmembrane proteins with distinct amino terminal, ligand binding, and transmembrane domains (Figure 2). They are expressed on postsynaptic membranes on neurons, and are expressed widely throughout the brain. Willardiine binds to glutamate receptors at the glutamate binding site in the extracellular ligand binding domain (Figure 2). Binding causes a conformational change that opens the receptor and allows for positively charged ions, Na+ and/or Ca2+ to enter the cell** (Figure 3). This depolarizes, and activates, the neuron, resulting in the firing of an action potential. The ion can also initiate a signaling cascade to activate different types of proteins that influence the cell, such as kinases or transcription factors.
**Passage of calcium through the AMPA receptor ("calcium permeability") is based on the presence of the edited GluA2 subunit (See: Glur2 RNA editing). The ion pore region of GluA2 mRNA is edited during translation to render it calcium impermeable in over 99% of AMPA receptors.
Like AMPA receptors, kainate receptors are tetrameric, transmembrane, ionotropic glutamate receptors on glutamatergic neurons. They have five subunits, divided into two main families: GluK1,2, and GluK5,6,7. An endogenous ligand, either kainic acid or glutamate, binds to the ligand binding site. However, unlike AMPA receptors, external ions also bind to kainate receptors at the ion binding pocket on the extracellular domain. When both the ligand and ion bind, the receptor undergoes a conformational change and the ion channel opens. This allows the flow of positive ions, such as sodium or calcium. The influx of positively charged ions depolarizes, or excites, the neuron (Figure 4). Like AMPA receptors, the permeability to calcium is dependent on the editing of receptor subunit mRNA. The rise and decay times of postsynaptic potentials is much slower in kainate receptors than AMPA receptors.
Kainate receptors are much less ubiquitously expressed throughout the brain and have a less significant role in plasticity than AMP receptors. In general, the function of kainate receptors is much less characterized than AMPA receptors. Interestingly, kainate receptors are expressed on the presynaptic membrane as well as the postsynaptic membrane. It is believed that presynaptic expression mediates the amount of neurotransmitter released from the cell (Figure 4). It is also known that kainic acid can induce seizures.
Both kainate and AMPA receptors are also affected by a variety of exogenous ligands, including willardiine. Only the (S) isomer of willardiine is a potent agonist of non-NMDA glutamate receptors. The precise binding has been characterized on cloned AMPA and Kainate receptors by analyzing the amount of displaced radiolabelled agonist (AMPA and kainate, respectively), which represents the binding affinity of willardiine to the receptor. (S)-willardiine displaces significantly less AMPA/kainate than its analogs, such as 5-Fluorowillardiine or 5-Iodowillardiine. Willardiine has the strongest binding affinity at AMPA subunit GluR4 and the kainate receptor subunit GluK5.
The displacement of glutamate by willardiine is primarily exothermic, with favorable binding enthalpy of -5±1 kCal/mol.
The structure and activity of willardiine were determined through elucidating the natural synthesis of willardiine and creating a synthetic mechanism of synthesis that can be used to create willardiine and its analogs to be studying in vitro and in vivo.
Synthesis The natural synthesis of willardiine and Isowillardiine from seedlings has been characterized (Figure 5). The synthesis consists of a free uracil ring at nitrogen being substituted for an alanyl side chain, derived from O-acetyl-L-serine.
The synthesis of willardiine has also been replicated in vitro, in which willardiine is synthesized from linear urea, which is made from a solution of acyl isocynate in Benzene with aminoacetaldehyde. Linear urea is cyclized to uracil when treated with aqueous alkali. Hydrolysis of uracil with Hydrochloric acid gives uracil-1-ylacetaldehyde. Hydrolysis of uracil-1-ylacetaldehyde with Potassium cyanide, Ammonia, and Ammonium chloride give the final structure of willardiine. This final synthesis of willardiine from uracil-1-lyacetaldehyde is carried out under the conditions of Strecker amino acid synthesis (Figure 5).
The lower binding affinity of willardiine for the AMPA and kainate receptors can be attributed to the unsubstituted carbon on position 5 of the 6-membered ring. Substitutions of large, electron-withdrawing groups at this position increase the affinity for kainate receptors but decrease the binding affinity for AMPA receptors. An example of the synthesis of one analog, 5-bromowillardiine, is shown in Figure 6.
Derivatives of willardiine have also been developed as Kainate receptor antagonists. These were synthesized through the addition of substituents to the N3 position of the 6-membered ring on the natural product willardiine (Figure 7).
Analogs Agonists The most common analogs of willardiine are also agonists of AMPA and kainate receptors. Each analogs differs in the substitution at the 5th position on the six-membered ring, and the most commonly studied analogs have a halogen at this position which is added through a Halogen addition reaction (Figure 6). The addition of a halogen affect the binding affinities and stability of the analogs. The analogs are more stable than unsubstituted willardiine and have better binding affinity for AMPA receptors, though the chemistry underlying the change in binding mechanisms remains unknown. 5-Fluorowillardiine 6-azawillardiine 5-iodowillardiine 5-bromowillardiine
Antagonists Derivative of willardiine have been developed as synthetic kainate and AMPA receptor antagonists (See: #Synthesis for specifics on development of antagonists). UBP282 (R = 4-COOH, (S)) UBP296 (R = 2-COOH, (R/S)) UBP302 (R = 4-COOH, (S)) These glutamate receptor antagonists all share a carboxylbenzyl substitution on the six-membered ring (Figure 7). UBP296 and UBP302 have been shown to have a 100-fold increase in binding kainate receptors over AMPA receptors, but UBP282 has competitive affinity for GluA2-containing AMPA receptors and GluK1-containing kainate receptors. These antagonists have therapeutic potential for a variety of neurological disorders characterized by aberrant activation of AMPA or kainate receptors (See: #Disease relevance). The pharmacokinetics of the compounds must be elucidated before they can be studied as potential therapeutics.
Pharmacokinetics It is crucial to understand the pharmacokinetics of any compound that has the potential to be developed as a therapeutic. These properties describe the ADME properties of the compound, which will determine the route of administration, dose, and potential adverse effects of a drug. The pharmacokinetic properties of willardiine have been studied using whole-cell recordings of mouse embryonic neurons. Willardiine desensitizes AMPA receptors with an EC50 of 44.8 uM, which makes it 4 times less potent that AMPA (EC50 = 11 uM), and 30 times less potent than its analog 5-Fluorowillardiine (EC50 = 1.5 uM).
Many of the pharmacokinetic properties of willardiine remain unknown due to a lack of in vivo or clinical research. Specific ADME properties have been predicted using admetSAR, a free tool that predicts properties of absorbance, distribution, metabolism, and excretion (Table 1).
The pKa of willardiine is approximately 10, meaning that willardiine is uncharged, or protonated, at physiological pH. +Table 1. Predicted pharmacokinetic properties of willardiinePROPERTYVALUESOURCEWater solubility13.2 mg/mLALOGPSlogP -0.7ALOGPSlogP -1.3ChemAxonlogS -1.2ALOGPSpKa (Strongest Acidic)9.76ChemAxonpKa (Strongest Basic)2.97ChemAxonPhysiological Charge0ChemAxonHydrogen Acceptor Count4ChemAxonHydrogen Donor Count2ChemAxonPolar Surface Area101.73 Å2ChemAxonRotatable Bond Count4ChemAxonRefractivity46.01 m3·mol-1ChemAxonPolarizability17.89 Å3ChemAxonNumber of Rings1ChemAxonBioavailability1ChemAxonRule of FiveYesChemAxonGhose FilterNoChemAxonVeber's RuleNoChemAxonMDDR-like RuleNoChemAxon
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